What’s That Brown Fuzzy Stuff on my Plum?
Protocol Sheet
- 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 it 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?
- 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 loosely in plastic bag with 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.
- 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.