DNA science extends cancer tool to crop diagnostics
- Early signs of fungicide resistance can now be detected in just hours
- The technology was developed through a GRDC-funded project by Dr Fran Lopez-Ruiz, who heads the fungicide-resistance group at the Centre for Crop and Disease Management at Curtin University in Western Australia, and research assistant Belinda Cox
New diagnostic technology borrowed from cancer clinics is making it possible to better manage the impact of fungicide-resistant powdery mildew on barley and wheat crops
Unlike in Europe, where barley varieties tend to be resistant to powdery mildew infections, growers in Western Australia are using susceptible cultivars because of their high yield potential.
Baudin, the variety preferred by Japanese maltsters, for example, is highly susceptible to powdery mildew.
The fungal organisms that infect barley and wheat are different (Blumeria graminis forma specialis hordei in barley and Blumeria graminis f.sp. tritici in wheat) and have a different distribution in Australia. The different geographical spread also reflects where susceptible varieties of wheat or barley have previously been grown widely, as the fungus carries over on stubble. Although fungicides help the grains industry manage disease risks, in recent years the cost and efficacy of this management strategy has been undermined by the emergence of fungicide-resistant strains of powdery mildew on both barley grown in the west and wheat grown in the east.
The situation has elevated the risks and costs associated with disease management on-farm, since the cheapest fungicides on the market have been the first to succumb to resistance.
What the situation has needed, according to Dr Fran Lopez-Ruiz, is the ability to provide growers with up-to-date information about the resistance status of powdery mildew strains in their paddocks – information that reveals which fungicides to avoid and when more expensive options would be worth the extra cost.
Dr Lopez-Ruiz heads the fungicide resistance group at the Centre for Crop and Disease Management (CCDM) at Curtin University in Western Australia. Recently, his team succeeded in developing a way to rapidly detect DNA mutations associated with fungicide resistance in crop samples of barley and wheat, even if present at extremely low levels.
The new test uses digital polymerase chain reaction (dPCR) technology that was first developed to detect cancerous mutations within human patient biopsies before they have proliferated and formed a large cancer mass.
“I have a clinical background and have worked in hospitals where I became familiar with dPCR and its ability to detect rare mutations within a mix of cells with different DNA subtypes,” Dr Lopez-Ruiz says.
“I thought dPCR could solve the analogous problem of detecting fungicide resistance mutations early in the season when they are present at low levels. Basically, this is technology that solves the problem of detecting a needle in a genetic haystack.”
Once leaf samples are received at the laboratory, the DNA is extracted in one pool that combines plant and fungal genetic material. The pooled DNA is then applied to a DNA chip so that one DNA molecule ends up within one of 20,000 micro-perforations.
Two small, marker-like pieces of DNA (called primers) are then used to target resistance mutations. If they are present, an enzyme amplifies the targeted DNA until there are enough copies to detect with a fluorescent probe.
“The whole process takes just four hours and is highly specific to the fungicide-resistance mutations,” Dr Lopez-Ruiz says.
“The method is also extremely sensitive, detecting resistance even if the pathogen’s DNA makes up just 0.1 per cent of the sample.”
He adds that the high specificity of his diagnostic probes owes a great deal to the availability of the complete genome sequence for the barley powdery mildew pathogen (Blumeria graminis f. sp. hordei). The genome sequence was made available in 2010 by an international team that included Dr Lopez-Ruiz, then at the Department of Disease and Stress Biology, John Innes Centre, Norwich, in the UK.
Subsequently protocols were worked up to detect fungicide-resistance mutations on wheat powdery mildew (Blumeria graminis f. sp. tritici).
From months to hours
What is remarkable about the dPCR breakthrough is that for the first time it is possible to know the pathogen’s population structure relative to fungicide resistance within hours. “Previously it has taken months and, on occasion, required samples to be sent to Germany, with results only available the following season,” Dr Lopez-Ruiz says.
Characterising powdery mildew strains has historically been exceptionally difficult because these pathogens are obligate biotrophs, meaning they cannot survive outside the plant host, including in the artificial media used in laboratories to propagate and analyse fungi.
“When we sequenced the genome we saw that the fungus lacks the genes needed to grow and metabolise independently of a host,” Dr Lopez-Ruiz says. “As such, this mildew will not kill its host but, rather, feeds so rapaciously that it causes devastating damage, including to grain quality.”
He hopes to make the new diagnostic capacity and its fast turnaround time available to growers who are affected by the emergence of fungicide resistance.
The rise of resistance
The selection pressure that led to resistance arose from the importance of the DMI (demethylation inhibitors) class of fungicides – not just in agriculture but also in human clinics. Growers first reported possible incidences of resistance in 2005.
In 2009 the former Australian Centre for Necrotrophic Fungal Pathogens, now CCDM, identified in Australia the same mutation associated in laboratories with mild fungicide resistance in other parts of the world, especially Europe.
“From 2009 to 2011, losses from barley powdery mildew suddenly jumped to an estimated $100 million just in WA due to DMI resistance,” Dr Lopez-Ruiz says. “In 2012 we isolated a new class of mutations that hadn’t been seen anywhere else in the world. These mutations were associated with extremely high levels of resistance to certain DMI fungicides.”
Strains bearing the more resistant mutations now make up the bulk of the barley powdery mildew population in WA, rendering some of the low-cost fungicides far less effective than in the past.
In eastern states, where barley powdery mildew traditionally is not an issue, the team first identified the strongly resistant mutation towards the end of 2015 in samples from Victoria, Tasmania and NSW. They have also identified, for the first time, the mutation associated with the milder, more common form of resistance in wheat powdery mildew.
So far the dPCR protocols have targeted the new, highly resistant mutations.
“Currently that amounts to nine distinct mutations. That would be fantastic because we could test any sample for any mutation in a quick and affordable way.”
That kind of capability would allow individual growers to know the particular mutations in their paddock and their abundance.
With knowledge about which fungicide-resistance mutations are present in paddocks comes the ability to use the most appropriate and effective management strategy.
“This is where we want to have an impact,” Dr Lopez-Ruiz says. “We want to support changes in disease management strategies. Existing approaches were set up decades ago based on now-outdated knowledge. With the availability of more powerful DNA-based diagnostic techniques, more effective management strategies
CCDM director Professor Mark Gibberd says that by thinking outside the box, the centre enables the rapid development and adoption of new technologies, such as dPCR. “By leveraging off discoveries in other areas of research, the centre provides innovations to the grains industry that help growers improve profitability and sustainability.”
More information (including sampling kits):
Dr Fran Lopez-Ruiz,
08 9266 1204,
GRDC Project Code CUR00023, CUR00016
Region Overseas, South, West