Research methods: Resistance is futile

Friends, family, and colleagues,

This post will discuss the main topic of my dissertation, fungicide resistance. To bring you up to speed on my pathogen, check out this publication

What is fungicide resistance?

Fungicide resistance or fungicide insensitivity is the phenomena where fungi can become less sensitive to fungicides after repeated exposure. In its simplest terms, when a fungal population (let’s say a population of Didymella bryoniae, my pathogen) is exposed to a fungicide, most of the fungi die off. However, as there is variation in the population and some of those variants have mutations that allow them to grow and reproduce in the presence of said fungicide.

If this sounds like something you’d read in Darwin’s “On the Origin of Species”, it’s because the process of fungicide resistance is a form of selection.

Why is fungicide resistance worth studying?

With many crops, there are multiple tools to combat pathogens. Growers can grow resistant cultivars, in which the plants are less susceptible to the pathogen. Growers can change the planting date in some cases to avoid the pathogen. They can also use treated seed to reduce the initial pathogen load.  With watermelon, there is no commercial resistance available. Growers in Florida can grow early, which helps avoid some pathogens. Seeds are regularly treated, but problems still persist.

The primary way that growers manage gummy stem blight is through the use of fungicides. This is problematic as fungicides can be expensive and resistance has been reported. It is important to characterize populations in the major growing regions to determine how much of a problem it really is.

So, why did you go to China to do this work?

We live in a global economy and 22% of all the watermelon seed imported into the US comes from China. In fact, if we look at the top 5 nations that export watermelon seed into the US, it accounts for nearly ¾ of all the seeds imported. The other four countries are Egypt (21%), Chile (17%), Peru (9%), and Israel (5%).

Given that D. bryoniae can be transported long distances on the seed, it is worth exploring potential risks of selecting for fungicide resistance in seed production areas as well as commercial production areas.

How is fungicide resistance measured?

There are many ways to measure fungicide resistance. For the purpose of my trip, we use a simple method called an amended agar assay. We grow the fungus on a nonamended plate and then we take a 7mm agar plug from that plate and place it on an amended plate.

All my agar plugs are placed face down so the fungi has to grow on the new plate

My fungus grows well on a media called Potato Dextrose agar, or PDA. Amended agar is exactly what it sounds; it is amending the agar with the fungicide. For this test, we add a specific amount of a single fungicide and compare its growth with an unamended fungicide.

There’s a lot of ways to measure, but I prefer a simple method. Typically, fungi will grow in a radial pattern, meaning that it will grow out from the center in a circle. I measure two points that are perpendicular (90^o from each other) at three time points. The time points for this fungus that work
best are 48, 72, and 96 hours. I’m very fortunate to have a fast growing fungus.

Control plate (right) with my 5 fungicide plates (center).
The top plates incidcate the this isolate is resistant to those fungicides while being sensitive to the bottom.

How much fungicide is used?

The rates of each fungicide that we’re testing varies. Fortunately, research has been completed to figure out a “baseline sensitivity”. The Fungicide Resistance Action Committee (FRAC) defines baseline sensitivity as:

“A profile of the sensitivity of the target fungus to the fungicide constructed by using biological or molecular biological techniques to assess the response of previously unexposed fungal individuals or populations to the fungicide.” 

This is simply how sensitive a population is to a specific fungicide. It has to be done for every fungicide I wish to test in order for my method to work. If it hasn’t been done, I get to calculate the baseline EC₅₀ with a lot of doses of the fungicide_. In my methodology, the fungal population is assumed to be sensitive to the fungicide at the EC₅₀. This is my discriminatory dose.

What is EC₅₀?

The EC₅₀ indicates the dose required to inhibit growth by 50%. So, for an isolate sensitive to a fungicide, say boscalid, the EC₅₀ is about 0.0034 mg/L. For every liter of agar I pour, there is 0.0034 milligrams of active ingredient in that agar. If the fungus is sensitive, it still grows (usually), but the growth is much smaller compared to a petri dish without the fungicide.

To give you an example of how small this is, take a typical 81mg aspirin tablet. Take that tablet and cut it in half. Repeat this halving for 10 more times. Now you have about 0.0034mg to put into a liter of agar.

I hope this was clear enough. If you have specific questions about fungicide resistance, feel free to leave them in the comments section. There are many different setups and I’ve given you an oversimplified method of what I am doing.

Mason