Appressoria Assay
How Do Fungi Infect Plants?
Microorganisms that cause diseases in living organisms are referred to as pathogens. In
general, fungi, bacteria, viruses and nematodes comprise the major groups of pathogenic
organisms commonly called germs. The most important pathogens in humans tend to be
viruses; for example those that cause colds and flues. In plants, fungi tend to be the more
important group of germs. There are over 10,000 different plant diseases caused by fungi
that cost farmers hundreds of millions of dollars a year in losses and chemicals to control
them. One of the more devastating fungal pathogens in the world currently is the one that
causes rice blast disease. This fungus, named Magnaporthe grisea, is responsible for
killing enough rice to feed over 60 million people a year. When we consider the fact that
rice is the most grown crop in the world and feeds over half the world population,
controlling the spread of this pathogen becomes particularly important. Because of this,
researchers worldwide are working to understand what genes the rice blast fungus uses
when it attacks a plant so that they can devise new ways of stopping it from killing rice
plants.
Conidium
In order to stop the fungus from
invading a plant, we must first
understand how it grows and
gets into the plant. Research
describing the life cycle and
process of infection for the rice
blast fungus is available. The
Appressorium
fungus is spread between plants
by the wind and physical
contact as asexual spores called
conidia. When a conidium
lands on a leaf surface, it sticks to the leaf surface and grows a germ tube also called a
hypha. After about 4-8 hours, the hypha stops growing and begins to swell at the tip.
The swelling continues as the water (turgor) pressure in the tip begins to build up. The
resulting swollen structure is called an appressorium. The purpose of an appressorium is
to build up enough pressure to be able to force a hyphal strand into the plant. Once inside
the plant, the fungus grows between the cells and begins to kill them. As the leaf tissue
dies, the fungus makes millions more conidia that are spread to new plants and the
process is repeated.
Plant leaves are covered with a
hard and waxy cuticle that is
difficult to penetrate. If fungi are
not able to breach the cuticle and
get inside the leaf, they can’t
access the needed nutrients to
colonize the leaf and cause disease.
Conidium
Germ tube
Appressorium
Penetration peg
Plant leaf
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The rice blast fungus uses the specialized infection structure (Appressorium: referred to
above) to get into the plant. Other fungal pathogens of plants use appressoria as well but
some simply rely on openings like stomata or wounds from insects.
The question we need to answer is; how does the rice blast fungus know that it is on a
rice plant and it is time to form an appressorium? It must get some cues from that plant
or its environment, but what are they?
The purpose of this exercise is to figure out what the fungus is sensing that tells it
form appressoria.
Activity:
For this activity, active cultures of the rice blast fungus will be supplied. The fungus
should stay within the school and at the end of the exercise all materials should be thrown
away, including the sent cultures. This activity will require the use of a microscope.
Day 1 – Each group will need the following:
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Petri dish culture of M. grisea
Plastic L-shaped spreading rod
Tube of sterile water
4 Plastic pipettes
One piece each of the surfaces to test: wax paper, gel bond (two
pieces), 4 glass slides, and cover slips.
Gloves
Tupperware container with lid and lined with damp paper towels
Marker
1. Line Tupperware container with damp paper towels.
2. With the pipette, transfer 10 drops of water from the tube provided onto the
fungal culture. The dark color of the culture is from the conidia.
3. Using the L-shaped spreader gently spread the water around the fungus while
rubbing the mycelia. This will knock the conidia free and suspend them in the
water.
4. Gently tip the petri dish on an angle and scrape the water to the edge.
5. Using a pipette, transfer two drops of the water containing the conidia from the
petri dish to the tube of water that you started with.
6. Gel bond has two sides to it that have different physical properties. We will test
both sides, so be sure to mark which is the top and the bottom sides of the gel
bond.
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7. Cut the gel bond and wax paper to the size of the glass microscope slide and place
them on the slide. Also place a cover slip on a glass slide. The gel bond should
be placed on two slides, one slide with the top side of the gel bond facing up and
one with the bottom side facing up. Put all the slides with the different surfaces in
the Tupperware container lined with damp paper towels.
8. On each surface being tested (wax paper, gel bond top, gel bond bottom, glass
slide, and cover slip) place two separate drops of water containing the conidia.
The conidia do sink in water, so be sure to shake the tube prior to removing the
drops. At the end, you should have a Tupperware container with 5 slides and two
drops per slide. The experiment should look like the illustration.
9. Being careful not to disrupt the drops on the slides, put the lid on the Tupperware
box and place it on a table in the room for 6 to 24 hours to allow appressoria to
form.
Wax paper
Glass Slide
Cover slips
Gel bond Top
Gel bond bottom
Paper Towel
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Analysis:
1. After a minimum of 6 hours,
gently place the glass slides under
the microscope and view the
spores. The spores should look
roughly like the figure to the right.
2. Count 20 random spores from
each spot and classify them as
forming appressoria or not.
Calculate the percent of spores
that form appressoria in each spot.
Conidia
Hypha
Appressorium
3. Look at the drop of water on each slide; write down what the drop looks like. For
example, is it rounded or does it spread out?
4. Compare each group’s results for consistency.
5. Answer these questions:
a. What basic difference is there between the surfaces that make the drop of
water look differently?
b . Does the difference in the surface affect if the fungus makes an
appressorium or not?
c. A rice plant blade (or a blade for grass for simplicity) mimics what
surfaces?
d. Write a conclusion to discuss what signal the fungus is sensing that tells it
to form an appressorium.
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