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Who Done It? Or what's that
brown fuzzy stuff on my plum? (Koch's
Postulates for Proof of Pathogenicity)
Lesson Plan
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?
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