Crime Scene Investigation (CSI) with Powdery Mildew Fungi.

This exercise can be used to stimulate the investigative nature of your students as they use forensic plant pathology techniques with powdery mildew cleistothecia (the sexual stage of powdery mildew fungi) to prove their innocence in a mock murder investigation. The exercise is suitable for grades 7 -12 and does not require the purchase or maintenance of special cultures, since cleistothecia produced by powdery mildew fungi are easy to find on plants in nature. Dissecting and compound microscopes are required.

Ruhl, G.. 2004. Crime Scene Investigation (CSI) with Powdery Mildew Fungi. The Plant Health Instructor. DOI: 10.1094/PHI-K-2004-0330-02

 

 

Purpose:

This exercise can be used to stimulate the investigative nature of your students as they use forensic plant pathology techniques with powdery mildew cleistothecia (the sexual stage of powdery mildew fungi) to prove their innocence in a mock murder investigation.

In order for students to truly understand the nature of science and technology, they must model the process of scientific investigation through inquiries. This lesson is suitable for grades 7 -12 and does not require the purchase or maintenance of special cultures, since cleistothecia produced by powdery mildew fungi are easy to find on plants in nature. Teachers will use leaves infected with powdery mildew fungi that they have previously collected.

Note: This lab works best in regions that experience cold weather for part of the year, since colder temperatures at the end of the plant's growing season encourages the production of cleistothecia (sexual stage of the powdery mildew fungi) on leaves. This lab may not work as well in areas where warm weather prevails year-round, since this type of weather promotes primarily the conidial (asexual) stage of powdery mildew fungi, with sparce to no cleistothecia production.

Background information:

CRIME SCENARIO:

An eyewitness thought he saw you and two others running from a wooded park where a murder took place. The police have brought you all in for questioning and during their investigation they notice that the leaf pieces stuck to all of your shoelaces have the same powdery-like appearance as the leaf pieces that were found on the murder victim's clothes. Although only circumstantial, the public is demanding the arrest of a suspect, and it appears that you are going to be arrested for murder unless you can prove them wrong. You must delve into the depths of forensic plant pathology and use diagnostic skills and tools to prove your innocence…that is, if you ARE innocent!

FORENSIC BACKGROUND INFORMATION:
Powdery mildew is a plant disease caused by a fungus that forms a whitish-gray coating of fungal hyphae (thread-like structures) and spores on the surface of leaves, stems, fruits, buds and flowers (Figure 1). Different genera of powdery mildew fungi can be identified most easily at the end of the plant's growing season when they reproduce sexually to form pinhead sized, black, spherical structures called cleistothecia (Figure 2). The distinctive 'arm-like' appendages that radiate from the surface of the cleistothecia and the presence of one or several spore-containing sacs called asci inside the cleistothecia help differentiate the genera (Figures 3, 4 and key to genera of powdery mildew).


Figure 1.


Figure 2.


Figure 3.


Figure 4.


Key to Genera of Powdery Mildew

Additional background information and color images are available in the K-12 Lab: Powdery Mildew Fungi: Classification and Ecology.

Powdery Mildew Fungi: Classification and Ecology

Purpose and Background Information

Purpose:

Powdery mildew fungi are ideal for use in a laboratory exercise on fungal classification, fungal spore types, host-parasite interaction during an ecology unit, or a study of host range. Students will discover how many different plant hosts they can find that are infected by the same genus of a powdery mildew fungus, or how many different genera of powdery mildew fungi can be found on the same plant host. This exercise demonstrates the diversity that exists within a fungal order. With a good collection of leaves infected with different powdery mildew fungi, students learn to use a written key and/or an illustrated key (or could even make their own key) to identify the powdery mildew fungus to genus. Since powdery mildew fungi reproduce by means of two spore types: asexual spores (conidia) and sexual spores (ascospores), discussions of the types of reproduction in fungi would be facilitated.

Background information:

Powdery mildews are one of the most common, conspicuous, widespread, and easily recognizable plant diseases. As a group, powdery mildew fungi infect many species of plants, including many trees and shrubs, numerous ornamentals, vegetables, cereals, grasses, and even weeds. However, individual species of powdery mildew fungi are usually very host specific. For example, the species of fungus causing powdery mildew on pumpkin is different from that causing the disease on roses.

Extensive losses in plant growth and crop yield occur annually due to powdery mildew. The primary sign of powdery mildew is grayish white, powdery blotches on leaves. Usually, powdery fungal growth appears first on the upper leaf surface (FIGURE 1). Eventually the entire leaf may become covered with mildew (FIGURE 2). Powdery mildew fungi also can infect other parts of the plant and may cause distortion and stunting of shoots, leaves (FIGURE 3), and flowers (FIGURE  4), and russeting on fruit (FIGURE  5). The type and extent of symptoms vary depending on the combination of powdery mildew fungal species and host plant species involved. Late in summer and fall, the sexual stage of many species of powdery mildew fungi, the cleistothecia, is visible as black or brown, pinhead-sized, spherical specks among the white to grayish mildew mycelium in the older infected areas on the leaves of many plants (FIGURE  6). (These are the leaves that should be collected and pressed flat between newspaper for storage and use in this laboratory exercise.)

Figure 1. 
The powdery mildew fungus, Uncinula necator, growing on the surface of a grape leaf. (Courtesy J. Schlesselman)
Click image to see a larger view.

 

Figure 2. 
This leaf is so severely infected that it is completely covered with powdery mildew. (Courtesy APS, Compendium of Stone Fruit Diseases)
Click image to see a larger view.

 

Figure 3. 
Distortion and stunting of young grape leaves infected by powdery mildew. (Courtesy J. Schlesselman)
Click image to see a larger view.

 

Figure 4.
Flowers distorted by the apple powdery mildew fungus, Podosphaera leucotricha, with white fungal growth on the stems of flowers in the center of the blossom cluster. (Courtesy K. D. Hickey)
Click image to see a larger view.

 

Figure 5.   
Net russeting of a Jonathan apple caused by powdery mildew. (Courtesy K.S. Yoder and K.D. Hickey)
Click image to see a larger view.

Figure 6.   
The white, brown, and black spheres are the cleistothecia of Uncinula necator at various stages of maturity (Courtesy W. Gärtel)
Click image to see a larger view.

The powdery mildew diseases of various crops and other plants are caused by many different species of fungi grouped into six main genera in the order Erysiphales, an order that includes a single family, the Erysiphaceae. The fungi causing powdery mildews are obligate parasites (or biotrophs), meaning that they cannot be cultured on nutrient media and require a living host to grow. These fungi reproduce by means of two spore types: asexual spores called conidia and sexual spores called ascospores.

The conidia are usually barrel-shaped or oval and are usually formed in chains (FIGURE 7) at the ends of specialized hyphae called conidiophores produced from the mycelium growing on the surface of a plant’s leaves, stems, flowers or buds. The combination of the mycelium, conidia and conidiophores gives the leaf surface a powdery appearance from which the name powdery mildew is derived. The fungi are spread when the conidia are carried by air currents to new plant surfaces. Under favorable conditions, the fungal spores germinate (FIGURE 8). Unlike most fungal parasites that invade plant tissues, most powdery mildew fungi grow superficially on top of the leaf surface (FIGURE 9). Fine, thread-like infection pegs penetrate the epidermal cells of the leaves and form haustoria (FIGURE 10). Haustoria are specialized hyphae for nutrient absorption from the plant cells. The plant is damaged by the loss of nutrients to the fungus, disruption of photosynthesis, and premature death of leaves or other infected plant tissues.

Figure 7.   
Conidia of Uncinula necator, formed in chains. (Courtesy W. Gärtel)
Click image to see a larger view.

Figure 8.   
Germinating conidia of Sphaerotheca pannosa on the surface of a rose leaf. (Courtesy of D. L. Coyier)
Click image to see a larger view.

Figure 9.   
Scanning electron micrograph of a colony of powdery mildew growing on the surface of a poinsettia leaf. (Courtesy G. Celio)
Click image to see a larger view.

Figure 10.   
Powdery mildew fungi absorb nutrients from the host plant through haustoria. Most powdery mildew fungi have simple globose haustoria (a), but species in the genus Erysiphe have more complex ones with fingerlike lobes (b). [(a) Courtesy G. Schumann; (b) Courtesy Plant Pathology Department, Washington State University 
Click image to see a larger view.

When environmental conditions or nutrition become unfavorable for growth (usually later in the growing season), the fungus shifts to the sexual stage and produces cleistothecia (sing. cleistothecium). The cleistothecia are closed, thick-walled, tiny, black, spherical structures (white to tan when young) that house sacs called asci (sing. ascus) (FIGURE 11 and FIGURE 12). The oval sexual spores, called ascospores, are produced within the asci. Fungi that produce ascospores in asci belong to the Ascomycetes, a fungal group that includes yeast, morels, and many plant parasites.

Figure 11.   
A cleistothecium of Uncinula sp. broken open to show the transparent saclike asci containing oval ascospores. Notice that the tips of the appendages look like hooks. (Courtesy B. Kendrick)
Click image to see a larger view.

Figure 12.   
Cleistothecia of Microsphaera spp.: a) Light micrograph of a Microsphaera sp. cleistothecium broken open to show several transparent saclike asci containing oval ascospores. Notice how the tips of the appendages are dichotomously branched. (Courtesy R. Wick) b) Scanning electron micrograph (500X) of a cleistothecium of Microsphaera alni. In this close-up view you can easily see the dichotomous branching at the tips of the cleistothecial appendages. (Courtesy M.F. Brown and H.G. Broztman)
Click image to see a larger view.

Cleistothecia have ‘arm-like’ appendages that radiate out from their outer surface. Inside each cleistothecium is a single ascus or many asci. Currently, the powdery mildew fungi are classified to genus based on the number of asci contained in the cleistothecium and on the morphology (physical appearance) of the hyphal appendages (arms) growing out of the wall of the cleistothecium (FIGURE 11, FIGURE 12, and Key to Genera of Powdery Mildew). Recently, Saenz and Taylor (1999) have proposed that powdery mildew genera be reclassified according to the phylogeny (an estimate of evolutionary relationships) inferred from the DNA sequence of the ribosomal ITS region and a number of morphological characteristics. If this occurs, the morphology of conidia and conidiophores will be used as the primary characters for classification.

 

Disclaimer: Powdery mildew fungi have long been classified to genus based on the number of asci contained in the cleistothecium and on the morphology of the hyphal appendages growing out of the wall of the cleistothecium. Recently, new research has resulted in the reclassification of many of these genera according to the phylogeny (an estimate of evolutionary relationships) inferred from the DNA sequence of the ribosomal ITS region and a number of morphological characteristics. In particular, the morphology of the conidia (asexual spores) and conidiophores (the structures that produce the conidia) is important in the new classifications. Despite these changes, this lab can still be used to teach students about forensic evidence, the use of identification keys, and morphological variation among fungal species.

 

MATERIALS AND METHODS

 

Collection Methods:

In late summer or autumn collect leaves with mature (black) cleistothecia on plants that show signs (pale, dusty-white coating on leaves) of powdery mildew. The cleistothecia should be visible to your eye without the aid of a magnifying glass. Asters, azalea, cherry, filbert, grape, horse chestnut, lilac, oak, phlox, rose, zinnia, evening primrose (Oenothera spp., often called sundrops), viburnum, wheat, willow and many other shrubs, trees, flowers, weeds and garden plants are good sources of cleistothecia.

Leaves exhibiting abundant cleistothecia are good specimens for teaching purposes. Infected leaves should be pressed flat between pieces of newspaper. Pressed, dried, leaf specimens can be stored in envelopes for use over many years. Label the envelope with the pressed leaves giving the name of the plant, the powdery mildew genus (you will need to identify the genus with the forensic key and diagrams), and location of collection, for future reference. 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 and Phyllactinia may both be found on oak. This is why it is important to label your envelopes by powdery mildew genus and not host plant.

Lab Preparation:

  1. Set out four empty envelopes and your envelopes of pressed, dried, leaf material infected with powdery mildew and covered with cleistothecia.
  2. Take three empty envelopes and label each of them "Leaf Material from Suspect's Shoes."
  3. Label the three envelopes #1, #2,or #3.
    Note: for a large class you may want to make duplicate envelopes of each number.
    (This exercise works best when students work in groups of three or four.)
  4. Remove several leaves from three of your storage envelopes that contain the pressed, dried, leaf samples of three different known genera of powdery mildew. Be careful to keep the leaves with each different powdery mildew genus separate from each other.
  5. Cut the leaves, one powdery mildew genus at a time, into smaller pieces. Clean the scissors before cutting the leaves of the next sample.
  6. Place the cut leaf pieces of each different known powdery mildew genus into one of the numbered envelopes (#1,#2 or #3) labeled "Leaf Material from Suspect's Shoes." Note: the number of cut leaf pieces must equal the number of groups in the class so that each group gets one piece of "Leaf Material from Suspect's Shoes."
  7. On a separate piece of paper (your key) write down the envelope numbers and the name of the known powdery mildew genera in each numbered envelope.
  8. Take one empty envelope and label it "Leaf Material from Murder Victim's Shoes."
  9. Choose one of the three known storage envelopes from which you previously removed leaves (so the Murder Victim sample will match only one of the Suspect samples) and remove several more leaves from that one envelope.
  10. Cut the leaves from that sample into smaller pieces and place them in the envelope labeled "Leaf Material from Murder Victim's Shoes."
    Note: the number of total cut leaf pieces in this envelope must equal the number of groups in the class so that each group gets one piece of leaf material from the "Murder Victim."
  11. Record on your key the name of the known powdery mildew genus that you placed in the envelope labeled "Leaf Material from Murder Victim's Shoes."

Lab Set-up:

  1. Place all of the labeled envelopes ("Leaf Material from Suspect's Shoes" and "Leaf Material from the Murder Victim's Shoes") on a designated table or lab bench.
  2. Place several black permanent markers or wax pencils on the tables for students to label their petri dishes.
  3. For each lab group, set up a work station area consisting of :
    • dissecting microscope
    • compound microscope
    • clear tape
    • two petri dishes
    • forceps
    • several microscope slides
    • water in dropper bottles.
      If supplies are limited, groups can easily share items.

Images for class use.

Procedures for students:

FORENSIC TOOLS:

You are a suspect in a murder investigation. You must attempt to prove your innocence and clear your name using dissecting and compound microscopes, clear tape, petri dishes, forceps, microscope slides, a water dropper and a forensic key with the special characteristics of powdery mildew cleistothecia appendages.

FORENSIC LAB PROCEDURES:

You may work alone or in a group of three or four.

  1. Collect and label forensic evidence.
    1. First, locate the numbered envelopes labeled "Leaf Material from Suspect's Shoes." (The suspect is YOU!)


Figure 5

 
    1. Choose one of the numbered envelopes labeled "Leaf Material from Suspect's Shoes" and using a forceps, remove a piece of the powdery looking plant material from the numbered envelope and place it in a petri dish you have labeled "Suspect."Record the number of the envelope on the petri dish and on your data sheet.
    2. Locate the envelope labeled "Leaf Material from the Murder Victim's Shoes" and using a different or clean forceps remove a piece of the powdery looking plant material from the envelope. Place the plant material in a petri dish you have labeled "Murder Victim." Note: Cleanliness is very important in this process of handling samples with forceps. There are several infamous cases of sloppy handling of forensic evidence that have led to false convictions.
    3. Take the labeled petri dishes with your pieces of plant material to your work station.


Figure 6

 
  1. Examine forensic evidence.
    1. Put a small drop of water in the center of a microscope slide.
    2. Examine the leaf sample labeled "Suspect." The black speck-like cleistothecia should be visible to your eye without the aid of magnification. Place the powdery looking leaf material peppered with black cleistothecia under a dissecting microscope, for increased visibility with magnification.


Figure 7

 
    1. Tear off a piece of clear tape that is approximately half the length of the microscope slide.


Figure 8

 
    1. While looking through the dissecting microscope, press the leaf piece firmly down with one hand. Using your other hand, hold onto one end of the tape and gently touch the rest of the clear tape on top of the black, speck-like cleistothecia. They should stick to the tape. Repeat this process several times on other areas on the leaf surface to gather more cleistothecia.
    2. Place the tape (sticky side down) on top of the water droplet on the microscope slide. Gently push down on the tape and smooth the tape over the water droplet so that it sticks to the slide. If necessary, more water may be added by squeezing the water dropper gently at the edge of the tape on the slide. The 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!
    3. 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 specks, then you have cleistothecia. If you do not see tiny black specks then remove the tape from the slide and repeat steps #4 and #5.
    4. Now 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).
  1. Identify forensic evidence.
    1. As you examine the cleistothecia with a compound microscope, look at the appendages on the cleistothecia. Carefully examine both the base and tip ends of the appendages on at least fifteen different cleistothecia to be sure you have seen the correct appendage type. This is necessary since tips break off easily, leaving only stringy looking "arms" that might lead you to a false identification. Compare the appendages 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 along with a drawing of the appendage type on the "Forensic Evidence Data Table" located on your Case Study Sheet.
    2. After observing the appendages, determine how many asci (spore-containing sacs) are in the cleistothecia by gently pressing down on the clear tape with the end of the forceps to break the cleistothecia open. It is helpful to look through the microscope at low power (100x) 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 Case Study Sheet. This information may be important in your effort to prove yourself innocent.

Repeat Sections B and C (Examination and Identification of Evidence) with the plant material in your petri dish labeled "Murder Victim" including the recording of results.

Forensic Key and Diagrams to the Genera of Powdery Mildew Fungi

Appendages coiled or hooked at tip - Uncinula
Appendages simple and straight with bulb-like base - Phyllactinia
Appendages branching dichotomously (antler-like at tip)
    Cleistothecium contains a single ascus - Podosphaera
    Cleistothecium contains several asci - Microsphaera
Appendages simple or irregularly branched, often entangled
    Cleistothecium contains a single ascus - Sphaerotheca
    Cleistothecium contains several asci - Erysiphe


Genera of Powdery Mildew Fungi
(Drawing courtesy of C.B. Kenaga, E.B. Williams, and R.J. Green)
  1. Record forensic evidence.

Locate the Forensic Evidence Data Table on the Case Study Sheet . Record and draw the type of appendages (arms) found on the surface of the cleistothecia (e.g. hooked tip; bulb-like base; antler-like dichotomous branching; or simple, string-like arms), the number of asci (one or several) contained within each cleistothecium, and the name of the powdery mildew genus.

Forensic Evidence Data Table

 

Characteristics of Cleistothecia

Powdery Mildew
Genus

Types of Appendages

Number of asci (sacs) per cleistothecium (one or several)

Murder Suspect (YOU) #______

 

 

 

 

 

 

Murder Victim

 

 

 

 

 

LESSON PLAN

Learning objectives:

To learn about a specific method of identifying fungi, using a written key and an illustrated key. Powdery mildew fungi can be identified to genus by the morphology (appearance) of the sexual stage (cleistothecia).

To learn about scientific investigation using forensic techniques.

Exercise description:

Plant leaves infected with powdery mildew disease are collected after the sexual stage, the cleistothecia, are visible on the leaf surface. Cleistothecia from leaf material are examined with a compound microscope to identify the type of appendages present. The fungi are identified to genus using the appendage type and the number of asci (spore-containing sacs) inside each cleistothecium using both a written and an illustrated key. The students, using forensic plant pathology techniques, must attempt to prove themselves innocent in this CSI lab. Images for class use are in the Supplementary Information and References section.

Time frame:

Infected leaves can be collected in late summer or autumn, pressed flat, and dried to preserve them until used. Fresh leaves also may be examined. Once you have a collection of powdery mildew infected leaves, the actual exercise takes little preparation and can be done at any time.

Study questions:

Forensic Conclusions: Innocent or Guilty?

  1. Compare the forensic plant pathology observations you have recorded for the two different samples. Are the appendages observed on the two different samples the same or are they different?

  2. If the appendages you observed on the cleistothecia found on the plant material taken from your shoelaces (murder suspect) are different from appendages on the cleistothecia found on the plant material taken from the murder victim, what does this evidence suggest?

  3. If the appendages on the cleistothecia found on the plant material taken from your shoelaces (murder suspect) are the same as the appendages on the cleistothecia found on the plant material taken from the murder victim what does this evidence suggest?

  4. Do you need an alibi as to where you were when the murder took place? Explain.

 

 

 

 

 

 

 

 

 

 

 

CASE STUDY SHEET

Investigators: _________________
___________________________
___________________________
Date: _______________________

CHAIN OF EVIDENCE TABLE

A. Collect and label forensic evidence.

Completed

1. Locate evidence (numbered envelopes) taken from suspects.

 

2. Obtain a sample of evidence from one of the numbered envelopes and place in container labeled with the word “Suspect” and the envelope number.

 

3. Locate evidence (envelope) from victim and place a piece of “Murder Victim” evidence in a separate container labeled “MurderVictim.”

 

4. Proceed to work station with labeled evidence from suspect and victim.

 

B. Examine forensic evidence.

Suspect

Victim

1. Water droplet placed on slide

 

 

2. Evidence examined with dissecting microscope

 

 

3. Small piece of clean tape obtained

 

 

4. Speck-like cleistothecia secured on tape

 

 

5. Tape pressed over water drop on slide

 

 

6. Check for successful evidence transfer to tape

 

 

7. Evidence examined with compound microscope

 

 

C. Identify forensic evidence.

1. Type of appendage (arm) identified with key, using compound microscope

 

 

2. Cleistothecia gently smashed to observe number of asci (sacs) inside.

 

 

3. Information and drawings recorded on Forensic Evidence Data Table below.

 

 


 

Forensic Evidence Data Table

 

Characteristics of Cleistothecia

Powdery Mildew
Genus

Types of Appendages

Number of asci (sacs) per cleistothecium (one or several)

Murder Suspect (YOU) #______

 

 

 

 

 

 

Murder Victim

 

 

 

 

Forensic Conclusions: Innocent or Guilty?

1) Compare the forensic plant pathology observations you have recorded for the two different samples. Are the appendages observed on the two different samples the same or are they different?

 

2) If the appendages you observed on the cleistothecia found on the plant material taken from your shoelaces (murder suspect) are different from appendages on the cleistothecia found on the plant material taken from the murder victim, what does this evidence suggest?

 

3) If the appendages on the cleistothecia found on the plant material taken from your shoelaces (murder suspect) are the same as the appendages on the cleistothecia found on the plant material taken from the murder victim what does this evidence suggest?

 

4) Do you need an alibi as to where you were when the murder took place? Explain.