Robert Koch was a German physician who carried out his work at a time (late 1800s) when the germ theory of disease was just gaining acceptance. Rigorous proof of pathogenicity was critical in order to show conclusively that microorganisms were the cause, not the consequence, of infectious diseases. Koch worked with a disease called anthrax that affected cattle and sheep. He developed several innovative techniques that enabled him to observe and grow the bacteria which were found in large numbers in animals suffering from anthrax. He was able to isolate the bacterium, grow it in pure culture, and demonstrate that it was capable of inducing the anthrax disease in healthy animals. In other words, he was able to demonstrate the cause-and-effect relationship between a specific bacterium and anthrax, the disease it caused. The steps in this procedure have been given the name Koch's Postulates and are still followed today in demonstrating microbial pathogenicity. The steps are as follows:

Step 1. ASSOCIATION: The suspected pathogen must be consistently associated with the diseased plant (or animal).

Step 2. ISOLATION: The pathogen must be isolated and grown in pure culture and its characteristics described.

Step 3. INOCULATION: The pathogen from pure culture is inoculated into a healthy plant of the same species or variety and it must produce the same symptoms and signs.

Step 4. RE-ISOLATION: The pathogen is re-isolated from the inoculated plant and its characteristics must be the same as the organism initially isolated in step 2.

In this exercise, Koch's postulates are carried out using the Sour Rot disease of oranges. This is a common soft rot disease caused by the fungus, Geotrichum candidum.

Oranges	70% ethanol, rubbing alcohol or 10% household bleach
Plastic bags	Petri plates containing potato dextrose agar (or other suitable medium)
Dissecting needles and forceps	Culture of G. candidum (available from Carolina Biological Supply)
Alcohol lamp

 

Inoculation Method: Surface sterilize oranges by rubbing with a tissue or paper towel moistened with alcohol or immerse oranges for a few minutes in 10% bleach. Blot dry on a clean paper towel. Flame sterilize a dissecting needle and allow to cool. Collect a small amount of the fungal culture on the needle and stab into the orange. Place the inoculated orange in a clean plastic bag, seal the bag, and incubate at room temperature. Within a week or so a soft, rotten area should develop around the inoculation point.

Isolation Method: The pathogen should be isolated from the margin of the infected tissue. This is the area where the soft, rotten tissue meets the firm, uninfected tissue. Surface sterilize the marginal region with alcohol. With flame-sterilized forceps, peel away a bit of the outer layer of the orange rind. Re-flame the forceps and take a small (about 2mm diam) piece of the rotten tissue from beneath the surface and place on the potato dextrose agar (PDA) within a petri dish. Put 3-4 pieces, well-spaced apart, in each dish. Incubate at room temperature. Over the next couple of days, white fungal colonies should develop around each piece of infected tissue. Transfer some of this fungal growth from the margin of a colony to a fresh plate of PDA to ensure that the culture is pure.

The Morphology of Geotrichum candidum: G. candidum, like most fungi, grows as a series of filamentous cells called hyphae. Its spores, called conidia or arthrospores, are produced by fragmentation of the hyphae. The hyphae and spores are white and appear colorless when viewed with a compound microscope. In order to observe the fungus in infected tissue, take a small bit of the rotten orange peel and place it in a drop of water. Put on a coverslip and gently press down to squash the tissue. Look for spores and hyphae among the dead plant cells.

Class Activity:

Step 1: Provide students with oranges showing symptoms of sour rot disease. Have students look at the rotten tissue with hand lenses or dissecting microscopes. The fungus can often be seen as a thin layer of white, slimy, hyphae and spores on tissue in advanced stages of decay. Use a toothpick or dissecting needle to transfer some of this material to a drop of water on a microscope slide and squash under a coverslip. Students should look for hyphae and spores.

Step 2: Once students have associated the fungus with diseased oranges they can follow the procedure outlined above to isolate the pathogen and grow it in pure culture.

Step 3: About one week later students can inoculate healthy oranges with the fungus they isolated from the diseased fruits. They should observe their inoculated oranges over the next week or so as the sour rot symptoms develop.

Step 4: The pathogen should be isolated from the inoculated oranges and its characteristics should be compared with the fungus isolated in step 2.

This exercise can also be carried out using a variety of other fruit rots caused by pathogenic fungi. The Brown Rot disease of stone fruits, caused by the fungus Monilinia fructicola also works quite well. Although Green Mold of oranges, caused by Penicillium sp. can be used, I avoid it because the fungus produces such an abundance of spores that a dropped petri dish or infected fruit can lead to lots of contamination problems in the lab.

I can usually provide the microorganism Geotrichumi candidum to Iowa teachers. Out of state teachers would have to get permits before I could send them this plant pathogen, even though it is common and not a risk. (Contact ebraun@iastate.edu).