Polymerase chain reaction (PCR) is a simple, widely used molecular biological technique which amplifies and detects target DNA and RNA sequences. Quantitative PCR (qPCR) uses fluorescently labelled DNA fragments, so that the amount of fluorescence detected during 2h amplification cycles can be used to quantify the amount of DNA in real time. New digital qPCR (dPCR) techniques offers even greater reliability and reproducibility in half the time. Both methods genetically fingerprint specific fungal pathogens in plant tissue, helping to determine fungicide choice and timing. They can also detect specific strains of fungi with modified fungicide sensitivity, aiding selection of effective fungicides and mixtures.
How it Works
In qPCR, the amount of amplified DNA is linked to fluorescence intensity using a fluorescent tag, which can be assayed in real time whilst amplification cycles are running. The more DNA template in the initial cycle, the quicker the detectable threshold of fluorescence is reached. At this point the absolute quantity of target DNA in the sample can be determined from a calibration curve of serially dilute standard samples with known concentrations. Once gene markers for specific fungi, or those with compromised sensitivity to specific fungicides have been identified, qPCR can be used to identify and quantify those markers in infected plant tissue. Battery operated mobile dPCR machines are now available, which can handle detection of up to 27 targets, and show great promise for field use.
Farmer / Agronomist Benefits
Identification of fungal crop infection during latent phases before visible symptom expression such as Septoria, permits better timed fungicide sprays. qPCR can also be used by plant breeders to help quantify resistance levels to specific pathogens, or in seed health testing programmes. The technique has also been invaluable in understanding mechanisms of fungal resistance to triazole and strobilurin fungicides, how these resistance genes build up in populations in response to crop management practices, and what new approaches can help reduce their spread.