Materials
Materials for collecting and drying leaves:
- Newspapers, old telephone books,
- Plant press, heavy books, or large catalogues
- Manila envelopes
- Paper for collection labels
Materials needed for this exercise are:
- Fresh or dried plant leaves with powdery mildew and mature (black to the naked eye) cleistothecia.
- Water in dropper bottles
- Binocular dissecting microscopes
- Compound microscopes
- Dissecting needles or toothpicks
- Pencils with good erasers
- Micropipets or Pasteur pipets and bulbs (optional)
- Microscope slides and cover slips
- Transparent tape
The following materials are helpful additions for working with dried leaves:
- Hot plate
- Large 800 ml beaker or coffee can
- Piece of screen to fit in the beaker
- Tong or forceps
- Envelopes
Methods
Collection Methods
Collect infected plant leaves in late summer or autumn that show signs of powdery mildew and mature (black) cleistothecia. Alternatively, assign groups of students to make their own collections. Check asters, beebalm (Monarda spp.), dandelions, lilac, phlox, rose, zinnia, evening primrose (Oenothera spp., often called sundrops), lawn grasses, wheat, crepe myrtle, cherry, apple, hawthorn, oak trees (red), willow, viburnum and many other shrubs, trees, flowers, weeds and garden plants for infected leaves. Look for a pale, dusty-white coating on leaves. The black, minute cleistothecia are generally visible to the unaided eye although a magnifying glass may help.
Leaves exhibiting numerous cleistothecia are good specimens for teaching purposes. If leaves are not used fresh, infected leaves should be pressed flat between pieces of newspaper placed in a plant press, or placed beneath some type of heavy object, e.g. large heavy books or catalogues. Use several layers of newspaper to absorb moisture from the leaves to prevent the leaves from becoming moldy while drying. You can also easily improvise a "plant press" by placing infected plant leaves between the pages of an old telephone book and then stacking a heavy book on top of the closed telephone book.
Dried leaf specimens can be saved in manila envelopes and re-examined over many years. Prepare a label on paper to store with the pressed leaves giving the name of the plant, date and location of collection, and the name of collector. For your own purposes of cataloguing and ease of choosing teaching material for labs, you may want to number your envelopes and keep a separate list of the specific genera of powdery mildew found to be associated with each plant collected. You will find that several different genera of powdery mildew will infect the same host type. For example, Microsphaera spp. and Phyllactinia spp. may both be found on oak; thus keeping good records is of great importance for lab set-up.
Preparation of dried material for class use
If leaves are dry, the appendages on the cleistothecia may break off when manipulated with a dissecting needle or toothpick. To prevent excessive breakage of appendages and to help insure a more successful lab, leaves may be softened by steaming. Using a hot plate, boil a small amount of water in a beaker or coffee can with a screen suspended over the water. Place the dry leaves in an open envelope. Using tongs or forceps, place the envelope over the screen to steam for 10 minutes. Using the tongs or forceps, remove leaves from the steam just prior to the beginning of class.
Collection of cleistothecia for microscopic observation
Place a leaf with cleistothecia under a dissecting microscope. While looking through the dissecting microscope, place a small drop of water on a group of cleistothecia. Still looking through the dissecting microscope, use a dissecting needle or toothpick to gently tease away several cleistothecia from the leaf surface into the water droplet. Then follow either step A, B or C:
A. Take a microscope slide and touch it to the water droplet on the leaf. Quickly flip the slide right side up and cover the droplet (which contains the cleistothecia) with a cover slip. If necessary, more water may be added by squeezing the water dropper gently at the edge of the cover slip.
B. Working under the dissecting microscope, draw up the cleistothecia suspended in the water droplet on the leaf’s surface with a micropipet or a Pasteur pipet and place them onto a slide and cover with a cover slip.
C. Using a piece of clear tape (smaller than the microscope slide), hold onto one end of the tape and smooth the rest of the tape (sticky side down) over the cleistothecia and water droplet. Slowly peel back the tape. You may also touch the tape to other areas on the leaf surface to gather more cleistothecia. Place the tape (sticky side down) on the microscope slide. If necessary, more water may be added by squeezing the water dropper gently at the edge of tape on the slide. Clear tape is an excellent tool for gathering cleistothecia and other fungal structures from a leaf surface. The tape also serves as a cover slip!
You can quickly check to see if you have successfully transferred cleistothecia to your microscope slide by placing it on a white sheet of paper. If you can see black pinhead-sized dots, then you have cleistothecia.
Place the slide on the stage of the compound microscope and examine the cleistothecia, first under 100x magnification, followed by 200x and 400x (if available).
Identification of genera
Look at the appendages on the cleistothecia. Carefully examine both the base and tip ends of the appendages on at least ten different cleistothecia to be sure you have seen the correct appendage type. Compare them to the written descriptions and/or the drawings in the keys included with this exercise. Decide what kind of appendages they are, and record this description on the data sheet included with this exercise.
Determine how many asci are in the cleistothecia by gently pressing down on the cover slip or clear tape with the eraser end of a pencil or a dissecting needle to break them open. It is helpful to look through the microscope at low power while doing this in order to see the asci pop out of the cleistothecia. Some cleistothecia may be immature and will not contain asci. Once you break open a mature cleistothecium, how many asci do you see? One ascus or several asci? Record your observations on the data sheet.
Results
A. Use the illustrated key to identify the powdery mildew to genus. Record data on paper or on classroom boards (see B).
B. Teachers may choose to list or illustrate the characteristics of the various genera separately on a sheet of paper and then have students create their own written or illustrated key. If identification and classification of several unknowns is done as group work, students can use classroom boards to share keys and results with other groups in a class discussion.
Observations of conidia (the asexual stage)
Observation of conidia is not necessary for identification of powdery mildew fungi, but may be of interest if the complete life cycle is discussed. Conidia are best observed on young, actively growing powdery mildew fungi. In the fall, leaves of Kentucky bluegrass growing in shady areas are a good source of new colonies. Fold a leaf in an area with powdery mildew, and observe the tiny chains of white conidia at the folded edge with a dissecting microscope (FIGURE 7).
 |
Figure 7. Conidia of Uncinula necator, formed in chains. (Courtesy W. Gärtel).
Click image to see a larger view. |
POWDERY MILDEW DATA RECORDING SHEET
|
Plant |
Characteristics of Cleistothecia |
Powdery Mildew Genus |
|
Appendage morphology |
No. of asci/ cleistothecium
(one or several) |
| |
|
|
|
| |
|
|
|
| |
|
|
|
| |
|
|
|
| |
|
|
|
| |
|
|
|
| |
|
|
|
| |
|
|
|
| |
|
|
|
How many different genera of powdery mildew did you find?
Was there more than one powdery mildew genus found on the same plant host?
Was the same powdery mildew genus found on different plant hosts?
