Research Methods: The Path of Least Resistance

Friends, family, and colleagues,

Forgive me for not writing sooner. I’m wrapping up the last of my experiments and unfortunately, that must take priority over blogging.

Measuring Pathogenicity

The last test I’ve been conducting is a test for pathogenicity. Pathogenicity is defined as an organism’s basic ability to parasitize a susceptible host. This can be broken down further by an organism’s “virulence” which is often characterized by having certain genes for enzyme or toxin production that makes it even better at parasitizing. For this test, we aren’t going that in-depth with our tests.

The Disease Triangle

Before I explain the experimental setup, I need to back up and explain an important concept in plant pathology, the disease triangle.

The triangle, as one would expect, consists of three sides. Each of these sides represents an aspect of a successful infection. In order to have infection, all three of the following aspects must be present.

Courtesy APS

First, a virulent pathogen must be present. This goes without saying, but if the pathogen isn’t present, then there is no disease. Second, a susceptible host must be present. Finally, the environment must be favorable for the pathogen to infect.

The disease triangle, while simple in its design, is incredible versatile for plant pathology research. Keeping two of the sides fixed allows researchers to conduct various experiments in a simple, straightforward manner.

For example, a plant breeder may screen for resistance by keeping the pathogen and favorable environment fixed which leaves the breeding lines to be the variable. Still another researcher interested in a fungal population can fix the host and the conditions and screen the population to gain insight on how pathogenic the population is on a susceptible host. These are simple examples and the variables that are changes can be much more subtle than what’s described here. The point is that the possibilities are nearly limitless.

If this disease triangle really interests you, I'd recommend visiting the APS website.

The detached leaf

For this experiment, I’m using a method called the detached leaf assay. Just as it sounds, it involves a leaf that has been detached from the plant. This method is fast, consistent and takes up very little space. Originally, the plan was to do this and a seedling test. However, the lab I’m in is not quite right for seedling tests. The growth chamber I’m in is set up for a process called tissue culture. So it was decided that a closed system like this would work better.

The leaf is exised (cut) from the plant and then is dipped in 75% ethanol to kill any insect pests. After a few minutes air-drying, the leaf is placed on a piece of filter paper which is then placed inside a glass petri dish. The paper is wetted with a couple milliliters of water and the pathogen is transferred from an agar plate. The petri dish is then sealed and measured at intervals of 48 hours for 6 days.

"Where are the controls?"

Whenever looking at an experiment, one question that scientists often ask is “what were the controls”. Without controls, there is nothing to compare the treatments to. In this experiment, I started out with a simple control, an untreated leaf. I followed all the instructions, except adding the pathogen. This allows me to see the effect that cutting the leaf and placing it into the chamber will have. A second control I used included a blank agar plug on an untreated leaf. This allows me to see any effect that the agar or anything in the agar may have on the leaf. Without these controls, it would be difficult to draw any useful conclusions from the data collected.


Rating system

The rating system is simply a 0 to 10 scale based on how much of the leaf tissue is necrotic (dead). A 0 is typical for our controls, as there’s nothing to kill the leaf and 10 is a really pathogenic isolate! I’ve added some photos so you see what some of the results look like.

No Pathogen? No problem!-Untreated plate (6 days)

Weakly pathogenic isolate (6 days)

Moderately pathogenic isolate (6 days)

Highly pathogenic isolate (6 days)

Quick Update-I'm coming home (soon).

I'm only here one more week. So I should be able to post a little more and catch up on a few more of my adventures while I've been in Nanjing. Stay tuned.

Mason

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

Watermelon industry day

Friends, Family, and Colleagues,

A few weeks ago, JAAS hosted an industry day for the watermelon industry in the Jiangsu province. It was similar to any industry convention you'd go to in the US, I've decided to make this a picture-heavy post to give you an idea of the varieties that are being produced on the other side of the world.

There were a LOT of free samples. It was mostly watermelon, but they did have muskmelon (which they call sweet melon over here).

Banner at the entrance

Opening remarks from the coordinators

Not all the watermelons were round...

There was a good turnout. Note the little kid pointing in my direction.
Americans are uncommon in this region of Nanjing.

This variety is translated to "ice cream" in English. Note the yellow and orange flesh. It was very sweet.

This was one of the trellis exhibits in the back of the building.
I'm here with Yuan, one of the students.

There is a tomato industry day this Saturday that I may check out. I don't know if there will be as many free samples. However, I could be mistaken, the Chinese love their tomatoes.

All is well over here.

Mason

Research Methods: Single Spore Isolation

Friends, family, and colleagues,

As part of my blog, I’d like to share with you, my readers, what I’m actually doing now that I’m settled into the institute. For the sake of my research, I won’t be discussing any results. That’s for once the seminars begin. For the sake of your sanity, I’m also keeping the methods very general, since I want this to reach to everyone. 

In plant pathology, it is said that the journey of a thousand experiments begins with a single spore. Ok, no one in my field says that, I just needed a cheesy introduction. 

The first step when I arrived to JAAS was to take the collection of isolates and culture them from a single spore. This technique is appropriately called the single spore isolation. This is done with most fungi for various reasons. The most important is uniformity. If you have a pure culture (that is, only one organism growing in your plate), there is still no way to ensure that the one organism is a single lineage or if it’s a diverse population. You could have 15 different individuals of the same organism in there, and that could cause some inconsistencies with experiments. Sometimes the fungi is grey or white, next time it’s green! 

This is NOT a pure culture. Some contaminants are clearly present

This is a pure culture. There's only one organism growing in there.
But is it all the same organism or a population of the same organism?

First step: Get spores

It may surprise you that in order to do a single spore isolation, you need spores. Now, that can be challenging for some fungi, since they don’t make spores. I had a labmate who worked on a pathogen called Sclerotium rolfsii that does not produce spores. There are some methods that can be used if that happens. However, that’s a whole different topic that I’m glad to avoid.

For my fungus, it produces two types of spores, asexual (called conidia) and sexual (called ascospores). Each kind of these spores is produced in a separate structure. Conidia are borne on a structure called a pycnidia, whereas ascospores are borne on either a perithecia or a pseudothecia (don’t get too hung up on the names, it has to do with the shape and structure of the fruiting body). For the sake of simplicity, conidia are the ones I want.

This a perithecium with ascospores emerging.

Pycnidia with conidia being released.
Each spore is a clonal copy of its parent

So how do I get these conidia?

The fungus has to be producing these pycnidia in order to get spores. There are a few methods that I use to get said spores. First, adding light to the growth chamber encourages the fungus to grow. Just as plants respond to light, so too will fungi. It isn’t essential for their growth, but it triggers certain responses, including pycnidiation. Adding in UV light also encourages pycnidia to form. Another method is to grow the fungus on a minimal media. If the fungus runs out of food, the next step is to make spores to find a better environment. 

I have spores! So now what?

So once we have spores, we have to count them. The best way is to take some of the spores from a petri plate and scrape them into some sterile water. At high enough concentrations, you can actually measure optical density (i.e. how “blurry” the spores are in water). However, there’s another way if fungus doesn’t produce billions of spores. There is a device called a hemocytometer (sometimes spelled with an "a" hemacytometer). Originally, it was produced to count red blood cells. It is also used to do sperm counts in both animals and humans. Fellas, if you ever go in for fertility testing, there’s a good chance that one of these will be used to assess your sperm.

Thanks Wikipedia!

So, the spore suspension is placed on the hemocytometer, and on the surface, there is a grid pattern. That grid pattern is viewed under a compound microscope. Within each grid is a known volume, typically 0.1µl. That’s one microliter, take your liter of cola and divide it by one million, Farva. We count the number of spores and from that concentration, we can adjust out suspension of spores to a desired amount. I’ll spare you all the math.

A typical grid pattern on a hemacytometer,
The red area (letter A for color blind) is 0.1ul in volume

I did the math! Now what do I do?

Once the spore suspension has been adjusted the suspension is then dropped onto another petri plate. For the sake of simplicity, I’ll go through an example. Say the suspension is adjusted to 1000 spores per 1ml of water, each one of those little droplets is going to be 1µl, which is 1/1000 of 1ml. In theory, there should be 1 spore within that aliquot. This is repeated several times as there is a lot of variance within that suspension. The conidia often come out of the pycnidia in a ribbon and are held together by a gel-like substance; adding a detergent reduces the gelling. Often, it’s called a “spore horn”. Also, this assay does not distinguish between viable cells and dead ones, so more chances are better chances. After the suspension has been dropped, the plate is left alone for 24 hours. 

If you mess with the spores, you sometimes get the horns.

I waited 24 hours, what’s the next step?

After the 24 hours, the plate is observed under the microscope and the spores will have germinated. Now comes the tricky part, an aliquot containing exactly one spore must be found. Given the variance, it’s not uncommon to find one aliquot with 6 germinated spores and the next aliquot to have none. After a bit of patience and luck, one perfect aliquot is found! We use a cork borer to carefully cut the agar chunk out of the original plate and that chunk is then plated onto a fresh, clean plate. After 48 hours, the single spore will have grown to the size where it is detectable to the naked eye. 

I successfully single-spored, now what do I do with it?

Now you can run all of your experiments! 

This is only the first step in the research journey. From start to finish it takes about 3 days to complete, assuming the culture is already creating spores. For the sake of consistency, nothing can be done before the single spore isolation, so it becomes a crucial task if working with fungi. I plan on generally describing some of the other experiments I’m running in the lab. I know I used a lot of technical terms, so feel free to ask for clarification in the comments.

I hope you found this interesting. If you have questions, leave them in the comments.

Mason