To study about the Koch’s postulates || plant pathology practical manual || BSc agriculture

 

Experiment no. 4

To study about the Koch’s postulates.

 

Koch’s postulates for plant pathogens

Introduction

 During the late 19th century a bacteriologist called Robert Koch laid down a set of rules for confirming that an organism is the cause of a disease. These are now known as ‘Koch’s postulates’.

When a plant becomes infected with a fungus (or any other disease causing microorganism), it is likely to become weakened and therefore more susceptible to infection by other microbes. So how do us plant pathologists work out what pathogen has caused a particular disease? We have to apply Koch’s postulates to the disease.

To determine Koch’s postulates:

(a) the organism must be consistently associated with the lesions of the disease;

(b) the organism must be isolated from the lesions and grown in pure culture;

(c) the organism from pure culture must be re-inoculated into the healthy host and must cause the same disease as was originally observed;

(d) the organism must be re-isolated into culture and shown to be identical to the organism originally isolated.

 Aim:

To demonstrate Koch’s postulates using apples infected with the brown rot fungus, Monilinia fructigena,.

 

Koch’s postulates: brown rot

To determine which microbe caused that fuzzy stuff on your apple you must:

 1. Describe the symptoms you see on the infected apple and isolate the suspected fungus pathogen responsible.

2. Isolate the fungus in pure culture (this means grow the fungus on its own, away from the host plant (apple) and without any contaminating microorganisms).

 3. Use the fungus that you isolated in pure culture to inoculate a healthy apple.

4. Record the symptoms that develop on the healthy apple following infection with the cultured fungus. Are your observations the same as recorded previously?

 5. Re-isolate the fungus and check that it is the same as observed initially.

Materials needed

1. Apples infected with Monilinia fructigena

2. Slides and coverslips

3. Cotton blue stain

4. Stereo & compound microscope

5. Imersion oil

6. Sterile scalpels

7. Potato dextrose agar (PDA) plus antibiotic

8. Fresh apples

Session 1

 You are provided with:

· An apple infected with the fungus Monilinia fructigena;

 · Three plates of Potato dextrose agar (PDA) supplemented with antibiotic (to control bacteria);

· Microscopes, slides, cover slips and cotton blue stain

 

Method:

1. Carefully record the symptoms of the disease. Examine the fruit externally. What do you see?

2. Slice through the infected apple. Are there any colour and texture changes in the infected fruit? Observe the symptoms using a microscope and compare them with a healthy apple (control).

 3. Using a sterile scalpel blade, carefully remove a pustule (containing reproductive structures of the fungus) from the infected apple and place it on a microscope slide. Place a drop of cotton blue stain onto the pustule and cover with a coverslip. Lightly press down the cover slip and use a circular motion to squash the pustule. This is best done using the end of a pencil. Take care not to crack the coverslip. Record your observations.

4. Using a sterile scalpel blade isolate small pieces of tissue from the edge of the infected apple and place one piece of tissue at the centre of each plate of PDA.

Session 3:

1. Compare the symptoms of disease in the inoculated apples with those recorded in session 1. Are they identical, or are there differences?

 2. You must now re-isolate the micro-organism from the apple inoculated in session 2, to show that it is identical to the organism originally isolated.

The plates will be incubated at 25o C and returned in session 4.

Session 4

. 1. Carefully examine the cultures.

2. Use the skills that you have learnt in the previous sessions to examine the cultures using microscopy.

3. Look for the characteristic spore bearing structures.