What’s That Brown Fuzzy Stuff on My Plum?
(Diseases are the Pits)
By: Jasalavich, C.A., and G.L. Schumann. 2001. Who Done It? Or what's that brown fuzzy
stuff on my plum? The Plant Health Instructor. DOI: 10.1094/PHI-K-2001-1128-01
Modified by: Rachel Hughes & Kirstin Bittel
Time:
These steps can be done at one-week intervals (3- to 4-week experiment), or
continued as cultures and fruit infections develop. It is helpful to wait for spore
production in both cultures and on fruit, so students can observe the characteristic
lemon-shaped spores.
If the shorter Germ Theory demonstration is done, the experiment will be completed in
five to seven days.
Preparation
Time:
Materials:
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stone fruit (peaches, nectarines, plums, cherries)
potato dextrose agar plates (3 options):
1) purchase pre-made plates (no additional materials needed)
2) make plates from purchased dehydrated potato dextrose agar medium
3) make plates from potatoes, dextrose and agar
For options (2) and (3) flasks, distilled water, autoclave, and Petri plates will be
needed.
dissecting needles
scalpels or single-edged razor blades
95% alcohol
matches
forceps
alcohol lamp or candle flame to sterilize dissecting needles and blades
10% (v/v) commercial bleach solution
sterile distilled water
paper towels
plastic box with lid
plastic bags with twist ties
dissecting microscope
compound microscope
microscope slides
cover slips
dropper bottle of distilled water
Abstract
Via laboratory experience, students use Koch’s postulates to determine the cause of disease in
stone fruits. Students explain both the root cause of disease in fruits as well as how the cause
was discovered.
Objectives
Students will be able to:
i.
Use Koch’s Postulates to determine that a specific organism is the root cause of a
specific disease and identify what Koch’s Postulates are within a protocol.
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ii.
Describe symptoms and signs of diseased fruit. Isolate fungal pathogen onto a nutrient
medium.
National Science Education Standard:
Content Standard G
 Science as a Human Endeavor
 Nature of Scientific Knowledge
Teacher Background
Both plants and animals can become ill due to pathogens. These disease causing pathogens
can be living organisms (bacteria, viruses, or fungi) as well as abiotic agents (for example air
pollution). Robert Koch (1843-1910) devised a scientific method to confirm causation of disease
by a microbe. His criteria are referred to as Koch’s postulates. Although still commonly used,
molecular methods have added a new dimension to disease identification. Some agents of
diseases that could not be identified by Koch’s postulates can now be identified through
molecular methods.
Monilinia fructicola, which causes brown rot of stone fruit (fruits with pits i.e. plums, peaches, etc),
is an easily available fungal pathogen that can be used for a simple demonstration of the Germ
Theory of Disease without the need for culture plates or several weeks of class time. A discussion
of Koch’s Postulates and their implications can be included. Infected fruit can be obtained at
supermarkets or farmers’ markets, or freshly infected fruit can be produced as described above.
Students can use spores from the infected fruit to inoculate healthy fruit. They should also
prepare disinfested, wounded fruit as controls. Both fruit should be incubated in separate plastic
bags for five to seven days at room temperature. When available, cherries can be used to provide
numerous fruit for less cost than a similar number of plums or other stone fruit. Take care to
select sound fruit for the experiment. Slightly under-ripe fruit are more likely to be disease-free.
For more detailed information see the website listed below.
http://www.apsnet.org/education/K-12PlantPathways/TeachersGuide/Activities/BrownRot/exercisepg1.htm
Related and Resource Websites
http://www.apsnet.org/education/K-12PlantPathways/TeachersGuide/Activities/BrownRot/exercisepg1.htm
http://www.apsnet.org/education/K-12PlantPathways/TeachersGuide/Activities/BrownRot/text/FIGURE13.HTM
Pre-Lab Preparation
Preparing Potato dextrose agar plates
1. Potato dextrose agar plates or potato dextrose dehydrated medium can be
purchased from Carolina Biological Supply Co. (http://www.carolina.com) and Ward’s
Natural Science Establishment, Inc. (http://www.wardsci.com). If the dehydrated
medium is purchased, directions for preparation of plates will be included.
2. Potato dextrose agar plates can be prepared from potatoes, dextrose, and agar
according to the following directions:
3. Boil 200 grams of peeled and sliced potatoes in 1 liter of water until the potatoes are
soft. Strain through cheesecloth and adjust the filtrate to 1 liter with more distilled
water.
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4. Add 10 to 20 grams dextrose and 12 to 17 grams agar. Autoclave 15 min at 121º C.
5. Pour autoclaved medium into sterile Petri plates. Makes approximately 40 plates.
Preparing 10% (v/v) bleach solution
 Mix one volume commercial laundry bleach, e. g. Clorox, with nine volumes of
distilled water.
Preparing fruit with brown rot for classroom use
1. For teachers who do not want to maintain or purchase cultures, it’s easy to find this
fungus just by buying stone fruit (peaches, nectarines, plums, cherries) and leaving
them at room temperature in a plastic or paper bag. They are often already infected,
and the infection will develop within a week, resulting in obvious brownish spores on
the fruit surface. Isolations from these fruits may be contaminated with bacteria and
other fungi, so a more successful lab for students can be accomplished by using
fruits that have been deliberately inoculated.
2. Prepare fruits about 1 week before they are needed. Disinfest (surface-sterilize)
firm, healthy stone fruit for 30 min. in 10% (v/v) bleach solution. Rinse with sterile,
distilled water.
3. Using a sterile dissecting needle, scrape spores from a culture of Monilinia fructicola
or a fruit with brown rot and stab each fruit four to six times.
4. Incubate at room temperature in a moist chamber (plastic box lined with paper towels
moistened with sterile, distilled water) with the lid not tightly closed. Check daily for
fungal development, which will vary with temperature in the lab and the
ripeness/susceptibility of fruit. Refrigerate the box of infected stone fruit if necessary
to preserve good disease development for student use (i.e. don’t let the brown rot
completely destroy the fruit).
5. Although the moist chamber as described above does not start out as a completely
sterile environment, because you do not sterilize the plastic box or the paper towels,
it does provide an environment adequate to favor the growth of the pathogen over
other organisms. A clean plastic box and fresh paper towels usually do not introduce
problems.
6. Infected fruit can be allowed to dry at room temperature to form a “mummy.” It will
probably be possible to use scrapings from the mummy to begin the disease again
when needed for another class.
Immediately before these lessons:
 Purchase healthy fruit to be used to test for pathogenic bacteria.
Activity
1. As students enter the room, have the following question on the board for students to
respond to: “Can plants get sick? Why or why not?”
2. Allow students a few minutes to record and share their thoughts with the rest of the class.
3. In their laboratory groups have students observe the diseased fruits you prepared earlier.
Ask them to describe the symptoms and signs of the diseased plums (or other stone
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fruit). Have them examine the suspected pathogen carefully both macroscopically and
microscopically and make notes about and drawings of what they see. They will want to
refer back to these recorded observations in later steps.
4. Ask students to consider how the diseased plum looks compared to a healthy plum. Is
something growing on the plum? Are some parts of the plum softer or firmer? What is
the color of the mycelium (hair-like, non-reproductive fungal growth) and spores?
Microscopically, what are the characteristics of the mycelium? Is it septate, i.e. does it
have internal cross walls that divide the hyphae into compartments? Or does the
mycelium just look like long tubes without any internal cross walls? Do you see any
spores? What are their shape, color, and size? Would you recognize this fungus if you
saw it again? (See below.)
5. Ask students how they would test to ensure that indeed it was the fungus that was
causing the disease. What variables and controls might they need to consider? Have
students write these tentative ideas down. Then ask students to talk within their groups
about what they think might be important factors to consider. As a class, discuss some of
the ideas that students think would be central to testing.
6. Explain to the students that Robert Koch (1843-1910) devised a scientific approach to
confirm causation of disease by a microbe. His criteria are referred to as Koch’s
postulates. [The students probably alluded to some of Koch’s postulates in their ideas
about how to eliminate the cause of the fungus as the root of the disease.]
7. Share with students Koch's Postulates:
I.
The diseased host is observed for signs of the causal organism and symptoms of
the disease; the causal organism is shown to be associated with all diseased
individuals.
II.
The causal organism is isolated into pure culture and described.
III.
This pure culture of the suspected pathogen is inoculated into a healthy host and
shown to cause the same disease symptoms and signs as originally observed in
Step 1.
IV.
The same causal organism is re-isolated into pure culture from the inoculated
diseased host and shown to be identical to the organism described in Step 2.
8. Explain to the students that they are going to use Koch’s postulates to determine the
cause of the disease in the stone fruit. (Step 4 can be eliminated if time is limited).
However, Koch’s postulates are not clearly identified in the protocol. As they carry out the
protocol they should identify which one of Koch’s postulates each step refers to.
Protocol
9. To isolate the probable pathogen on a nutrient medium, e.g. potato dextrose agar (PDA):
a) Cut four small (2 mm x 2 mm) pieces of infected fruit tissue.
b) Disinfest briefly by immersing the four pieces of tissue for 15, 30, 45, or 60 sec in 10%
(v/v) bleach solution.
Note: This is done to remove any surface contaminants without killing the pathogen
deeper in the tissue. Since it is not known exactly how long this takes, several
different times are chosen to ensure a successful isolation of the pathogen. You want
to disinfest the tissue of any contaminating organisms, but not kill the fungal
pathogen.
c) Sterilize forceps by briefly passing them through a flame and allow them to cool. Using
sterile forceps, remove the tissue from the bleach and blot dry on a paper towel.
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d) Place each piece on the surface of the PDA agar in the Petri plate. Minimize the time
that the medium in the plate is exposed to possible contamination from spores in the
air.
e) Incubate at room temperature for five to seven days.
10. Describe the isolated pathogen in culture both macroscopically and microscopically. Record
these observations as words and drawings. Do you think this is the same organism that you
observed on the diseased fruit in Step 1?
11. Use the isolated pathogen to inoculate healthy plums as follows:
a) Immerse 2 healthy plums in a 10% (v/v) bleach solution for about two minutes. This
disinfests the fruit of any surface contaminants. In the original fungal isolation onto
PDA, small pieces of cut fruit were placed in the bleach solution for a shorter time to
avoid killing the pathogen deeper in the tissue. The 2 minute time for whole fruits can
be used because the intact skin of the fruit protects the inner flesh from the chlorine.
Remove and dry with paper towels. Make a V-shaped cut with a sterile blade on the
surface of the first plum. Place loosely in a plastic bag with a moistened paper towel,
close with a twist-tie and label. This is the control plum.
b) Repeat with the second plum with this change: inoculate the wound with spores from
your isolate using a sterile dissecting needle.
c) Incubate for one week and record your observations of any symptoms and signs that
develop on each fruit.
12. Koch's postulates require that the pathogen be isolated from the inoculated fruit-as in Step 9
(step 1 on the student protocol sheet) to determine if it is the same organism that was originally
observed on the first diseased fruit.
13. Once students have had time to allow the disease to grow on the healthy fruit and confirm the
cause of the disease, bring the class together to discuss the results and to identify how they
thought Koch’s postulates were addressed in the protocol.
14. Students may wish to design experiments to further investigate factors that affect disease. For
example:
1) What is the effect of temperature on infection and disease development?
Inoculated fruit can be placed at room temperature and in a refrigerator for a simple
comparison. Why do we refrigerate most fruits and vegetables after purchase?
2) What is the effect of wounding on infection and disease development?
Spores can be applied to wounded and non-wounded fruit for a simple comparison. What
are some potential sources of wounds in commercial fruit production, harvest and
shipping?
3) What is the host range of Monilinia fructicola?
Students can bring in healthy fruits and vegetables for inoculation to determine which
ones are susceptible to brown rot. Stone fruit are the common hosts of this fungus, but
ripe apples and pears sometimes develop the disease. Which species develop brown rot
when inoculated with Monilinia fructicola and which ones do not develop the disease?
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Embedded Assessment
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o
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Are students able to identify variables and controls that they should consider when attempting
to identify the cause of disease?
Can students identify which steps in the protocol are associated with which of Koch’s
Postulates?
Are students able to isolate the pathogen and if not, are they able to identify what might have
gone amiss in their procedures?
Homework
In their science notebooks, have students write a reflective conclusion. What did they learn?
What new questions do they have? How does the lab connect to “real life?”
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