Learning Objectives
To learn how to establish that a specific organism is the cause of a specific disease. The criteria used are called Koch’s Postulates for proof of pathogenicity, an important component of the Germ Theory of Disease.
Exercise Description
Students will learn about Koch’s Postulates by actually using them. They perform each of the required four steps to show that the brown fuzzy fungus they see growing on stone fruit (plums, peaches, nectarines, cherries) is what causes them to rot. Although sterile technique is not used throughout the protocol, it is adequate to favor the growth of the pathogen over other organisms. The fungi that cause brown rot of stone fruit are not human pathogens.
Time frame
Pre-class by teacher
1) Buy stone fruit and let sit at room temperature for a few days to one week to allow fungal development from naturally occurring infections. Although most of the fruit will develop brown rot, look for Italian or prune plums with flecks. These flecks are quiescent brown rot infections (Figure 13). Stone fruits with brown spore masses on their surface probably also have brown rot.
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Figure 13. Quiescent infections caused by
Monilinia fructicola shown as small flecks on prune fruit. (Reprinted by permission from Ogawa and English, 1991). Click image for a larger view. |
2) Inoculate fruit for class use with brown spores from the above fruit and incubate for few days to one week, i.e., until you see symptoms and signs beginning to develop.
3) Right before the first class period, buy healthy-looking stone fruit (no disease symptoms or signs) to be used for inoculations.
In class by students:
Describe symptoms and signs of diseased fruit. Isolate fungal pathogen onto a nutrient medium.
Examine plates and describe isolated pathogen. Inoculate healthy fruit with isolated pathogen.
Allow inoculated fruit to incubate and isolate pathogen from developing infections.
Examine fungal isolate from inoculated fruit.
These steps can be done at one-week intervals (3- to 4-week experiment), or continued as cultures and fruit infections develop. It is helpful to wait for spore production in both cultures and on fruit, so students can observe the characteristic lemon-shaped spores.
If the shorter Germ Theory demonstration is done, the experiment will be completed in five to seven days.
Study Questions
- What is a pathogen?
- What is disease?
- What is a symptom?
- What is a sign?
- Why do fruit rot?
- What are Koch's postulates and what is their significance?
- What are some complications to the application of Koch’s Postulates to various diseases?
Study Question Answers
1. What is a pathogen?
A pathogen is an organism that can cause disease, e.g., fungus, bacterium, nematode, virus, or an abiotic agent, such as air pollution.
2. What is disease?
A disease is a harmful disruption of the normal physiology, growth and development of an organism by a causal agent.
3. What is a symptom?
A symptom is the reaction of a host to infection by a pathogen. Wilt, yellowing, leaf spots, and brown dead tissue (necrosis) are all examples of symptoms that may indicate disease in plants. Fever, sneezing, and a runny nose are symptoms of the common cold, a disease of humans.
4. What is a sign?
A sign is the physical presence of a pathogen or part of a pathogen on or in a diseased organism. For example, fungal hyphae and spores present on a rotting plum are signs of brown rot of stone fruit. The large numbers of bacilli present in the blood of an animal that has died from anthrax are a sign of anthrax.
5. Why do fruit rot?
Students should discuss why they disinfest their fruit with the 10% bleach solution before inoculating them. The purpose of this is to kill any pathogenic spores that are on the surface of the fruit. Most students are surprised that a wounded fruit can be maintained in moist conditions in a plastic bag for a week without rotting. If they believe in the Germ Theory, they should consider the source of the pathogens that cause their fruits and vegetables to rot after purchase. They should also consider why the fruit were wounded before inoculation (see Additional Experiments below).
6. What are Koch's Postulates and what is their significance?
Koch's Postulates are:
a) Observe and describe the signs and symptoms of disease.
b) Isolate the suspected pathogen.
c) Inoculate a healthy host with the isolated pathogen to see if you get the same signs and symptoms produced as in (a).
d) Isolate the pathogen from the inoculated host (c) to see if you get the same pathogen as observed on the original diseased host.
Koch's Postulates are significant because they provide us with an experimental way in which to identify and confirm that a specific pathogen causes a specific disease. A diseased organism may have many other microorganisms present on its surfaces or inside its organs and tissues besides the microorganism that is actually causing the disease. Therefore, we need rigorous criteria to prove which specific microorganism is the culprit. Students can discuss why it is not sufficient to simply inoculate a healthy fruit with spores directly from an infected fruit.
7. What are some complications to the application of Koch’s Postulates to various diseases?
Koch’s Postulates were an important accomplishment in the early days of the Germ Theory. We now know that microorganisms do not cause all diseases. Consider arthritis, diabetes, and nutrient deficiency in humans. Plants also can suffer from nutrient deficiencies as well as the effects of air pollution and other environmental stresses.
Even with diseases that are caused by microorganisms, it is not always easy to apply Koch’s Postulates. Some pathogens are obligate pathogens and cannot be grown in culture. Common examples among plant pathogens include rust fungi, powdery mildews, downy mildews, phytoplasmas, nematodes, and viruses. There also are many obligate parasites among animal pathogens. Modifications of Koch’s Postulates are necessary to separate the pathogen from the infected host before inoculating a healthy host. Inoculation of a healthy host plant also is not always as easy as with brown rot. For example, phytoplasmas and some viruses require an insect vector for inoculation. Simply applying the pathogen to a wound on the plant will not result in infection.
Students also may want to discuss some of the ethical aspects of applying Koch’s Postulates to human diseases. The procedure requires inoculation of a healthy individual. This has led to controversy about the cause of certain diseases (such as HIV and AIDS) because we do not deliberately infect humans with isolated pathogens. Koch himself did not inoculate humans to prove the cause of tuberculosis, but used other animals known to be naturally susceptible to the disease as model systems. He isolated the causal bacterium of tuberculosis from the lungs of people who had died of the disease and inoculated healthy guinea pigs and rabbits which subsequently developed the disease. He was able to isolate the causal organism from the dead animals and show that it was the same as the one he had originally isolated from people.
Additional experiments:
Students may wish to design experiments to further investigate factors that affect disease:
1) What is the effect of temperature on infection and disease development?
Inoculated fruit can be placed at room temperature and in a refrigerator for a simple comparison. Why do we refrigerate most fruits and vegetables after purchase?
2) What is the effect of wounding on infection and disease development?
Spores can be applied to wounded and non-wounded fruit for a simple comparison. What are some potential sources of wounds in commercial fruit production, harvest and shipping?
3) What is the host range of
Monilinia fructicola?
Students can bring in healthy fruits and vegetables for inoculation to determine which ones are susceptible to brown rot. Stone fruit are the common hosts of this fungus, but ripe apples and pears sometimes develop the disease. Which species develop brown rot when inoculated with
Monilinia fructicola and which ones do not develop the disease?
