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Purpose and
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Materials and
methods

Lesson plan

Supplementary
information and References

Diagrams
for class use

• 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

1) Potato dextrose agar plates

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.

Potato dextrose agar plates can be prepared from potatoes, dextrose, and agar according to the following directions:

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.

Add 10 to 20 grams dextrose and 12 to 17 grams agar. Autoclave 15 min at 121º C.

Pour autoclaved medium into sterile Petri plates. Makes approximately 40 plates.

2) 10% (v/v) bleach solution

Mix one volume commercial laundry bleach, e.g., Clorox, with nine volumes of distilled water.

For teachers who do not want to maintain or purchase cultures, it’s often 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. Infected fruit may be more difficult to find in arid regions or in dry years. 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 fruit may be contaminated with bacteria and other fungi, so a more successful lab for students can be accomplished by using fruit that have been deliberately inoculated.

Prepare fruit 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.

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.

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).

Infected fruit can be allowed to dry at room temperature to form a “mummy.” It will probably be possible to use scrapings from a mummy to begin the disease again when needed for another class.

Koch's Postulates for Proof of Pathogenicity

1) Describe the symptoms and signs of the diseased plums (or other stone fruit). Examine the suspected pathogen carefully both macroscopically and microscopically. Make notes about and drawings of what you see. You will want to refer back to these recorded observations in later steps.

How does the diseased plum look 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 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 is their shape, color, and size?

Would you recognize this fungus if you saw it again? (See below.)

2) 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 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.

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 min 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 in a plastic bag with some air so the plastic doesn't cling to the fruit too much. Add a moistened paper towel and close with a twist-tie. 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 should be isolated from the inoculated fruit (as in Step 2) to determine if it is the same organism that was originally observed on the first diseased fruit. This step may be skipped if time is limited.

Monilinia fructicola, which causes brown rot of stone fruit, 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 perform Step 3 (above) using 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.