How do we use genomics in our research on regulatory plant pathogens?

August 2019 | Canadian Food Inspection Agency | by Guillaume Bilodeau and Emily Giroux

My kids always ask me, “Dad what is your job? What do you do at work?”

My response is simple, “I work with DNA. I use DNA to identify diseases affecting plants.”

“DNA as in Mr DNA in the Jurassic Park movie?!” they ask.

“Yes, like Mr DNA. I try to find differences in the DNA of organisms so I can develop markers to help us detect particular plant disease-causing fungi.”

The Canadian Food Inspection Agency (CFIA) protects Canada’s food, animals, and plants - that includes making sure our plants are healthy and free of harmful pests. It’s much easier and cost effective to control harmful plant pests before they become established. This makes early detection of non-native plant pests a key part of pest management efforts. Because invading species may first occur at low densities, we need detection methods that can spot them even when present in low numbers. This can be a challenge when the pests that cause disease—usually bacteria or fungi—are hard to grow in the lab, or look like harmless organisms.

In the past we depended on morphology to classify and group similar living things. This approach requires lots of time and mastery and often isn’t enough to tell species apart. We can overcome these issues using molecular techniques that identify organisms based on their genetic information. More and more we use genomics to design molecular markers to help us detect and track plant pests.

Environmental samples can have rich communities. Hundreds of different species may be present in a single soil sample. In the lab, we make use of the fact that the genome of every species is different. Consider a leaf sample taken from an infected tree. We crush the leaf sample to break open the cells and release the DNA. Since the DNA in this sample comes from both the plant and its pests, we need to distinguish which pieces of DNA belong to which organism. Small differences in the DNA sequence of a shared gene region can serve as barcodes that can inform us of the species we have in a sample.

How we proceed depends on our aim and if we have the genome sequence of the target organism and a barcode for it. If a good enough barcode exists, we can go straight to metabarcoding. We start by extracting DNA from the sample and make copies of the short barcode/marker regions. After we isolate these regions, we amplify them using PCR to increase their signal before sequencing. The amplified DNA sample is passed on to a high-powered machine called a Next-Generation Sequencer that can sequence millions of DNA molecules at a time. The sequences detected by the sequencer can then be compared to thousands of others in a barcode database to find a match. Because this approach uses a common DNA region, we can analyse many species in a sample at the same time.

Thus, an organism’s entire genome (whole genome), or genomes of many organisms (meta genomes), in a DNA sample can be sequenced in a matter of hours. These innovative technologies can handle large numbers of samples at the same time and create huge volumes of genomics data. By mining the genomes of related pests and comparing them to one another, we can design tests that distinguish cryptic species based on their DNA. The genetic information gathered from environmental samples can further allow the CFIA to trace the path of plant sicknesses in nature.

If we don’t have the genome sequence or a barcode for a pest we want to detect, we need to grow it in a lab and extract its DNA so we can sequence its entire genome. Then we use comparative genomics methods to help us find DNA regions with genetic differences that can distinguish our target organism from its relatives. These have potential for being suitable markers. We can likewise add the markers to our reference database. That way, when we scan biological communities or run quick environmental surveys to find disease hotspots and for monitoring, we can detect the pest.

Once we’ve optimised and verified our custom barcodes, we send them on to the CFIA’s plant diagnostic labs for testing samples collected from various plant products. Barcodes that can pick up on trace amounts of DNA of specific target pests make it easier for diagnostic labs to test samples. At the CFIA we use genomics often. For example, we recovered genomic information from insect traps used in our annual insect survey. By using meta-barcoding to analyse the DNA extracted from these traps, we could keep an eye out for plant pests that travelled on the insects.

Scientific advances have come a long way. As genomic technologies improve, so will monitoring sensitivity and response timeliness to a detected plant disease. New genomic instruments as small as USB thumb drives can now carry out the same level of sequencing as did earlier instruments that were the size of refrigerators. Working with “Mr DNA” continues to become easier, faster and more portable. This challenges us to develop the best tools for the CFIA to use while responding to its mandate of protecting plant and animal health, and food safety.

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