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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:

Protocol Sheet
• 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.
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-2PlantPathways/TeachersGuide/Activities/BrownRot/text/FIGURE13.HTM

Quiescent infections caused by Monilinia fructicola shown as small flecks on prune fruit.
(Original image belongs to Ogawa and English, 1991).

 

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.
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 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

1. 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.

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.

2. 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?


3. 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.

4. 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.

5. 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.

6. 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?

Embedded Assessment

  • 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?”

Embedded Assessment

 

 


PULSE is a project of the Community Outreach and Education Program of the Southwest Environmental Health Sciences Center and is funded by:


an
NIH/NCRR award #16260-01A1
The Community Outreach and Education Program is part of the Southwest Environmental Health Sciences Center: an NIEHS Award

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Supported by NIEHS grant # ES06694


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Last update: November 10, 2009
  Page Content: Rachel Hughes
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