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