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Sheet 18 
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Robert Koch was a bacteriologist who lived from 1843 to 1910. In his work on diseases, he was trying to establish whether a microbe, obtained from a diseased patient, was in fact the cause of the disease. This led him to formulate ‘Koch’s Postulates’ - a set of conditions which need to be fulfilled to establish which organism is causing a particular disease. Many plant pathogens are safe to use in a school or college laboratory (but simple safety precautions must be observed - see safety notice below). If you attempt to isolate a pathogen from infected fruit, vegetable, or other plant tissue, you are likely to obtain a plate with several organisms growing on it. This is because, once a plant becomes weakened by disease, secondary saprophytic organisms often invade. So, when identifying which is the disease-causing organism, the first step is to demonstrate Koch’s Postulates. To demonstrate Koch’s Postulates, you must do the following. (i) Describe and record the symptoms shown. (ii) Isolate the suspected pathogen from the infected plant material and establish a pure culture. (iii) Use the pure culture to infect new plant material. (iv) Describe and record the symptoms shown by the new plant. Check that these are the same as your original observations. (v) Re-isolate the organism. Check that this is the same as that isolated previously. This protocol allows students to demonstrate and test Koch’s Postulates, using apples infected with the fungus Penicillium expansum. The required experimental work extends over 3 to 4 weeks, but the essential steps can be demonstrated in a single practical session if students are provided with cultures and infected fruit which represent different stages in the sequence of steps. Preparation for the practical work Agar plates and apples need to be inoculated about one week before the first practical session and incubated at 20 to25oC. When inoculating agar plates from infected apples, it is best to push the piece of infected tissue right into the agar jelly in the centre of the plate. Plates should be inoculated the right way up so need to be poured in a way that gives very little condensation. Each group is provided with 4 apples
Each group also has
In carrying out the steps in the protocol, always work with the ‘control’ apple first, to avoid cross contamination.
Penicillium expansum does not produce penicillin but anyone who suffers from allergies should be especially careful and must wear a face mask.
Note 2. Examine the infected apple (externally). Compare with the control. Apple 1 has been inoculated with Penicillium. Apple 2 is the control (see 2) Note 3. Examine the infected apple (internally). Compare with the control. Cut the infected and the control apples in half, through the inoculation point. Describe the type of rot you see. Note
5.
After 7 days you should have one or more fungal colonies on your
plate. Infect a new apple using the culture on the plate (from step
4)
Now check through to see if you have demonstrated Koch's Postulates (see the statements at top of this page). If your answers to numbers (iv) and (v) are 'yes', then you have isolated the organism which is causing the disease. Examining the fungus under the microscope
Notes for teachers Media recipes for fungal practicals Multi-purpose media for fungal isolation and growth. All media autoclaved at 15 p.s.i. 121oC for 15 mins. Malt agar Malt extract 20 g, agar 20 g, distilled water to 1 dm3 You can use malt extract without additives from the chemist, but will need to adjust the pH to 6.5 using NaOH. Chemicals and powdered media may be bought from: • Sigma-Aldrich Co Ltd 0800 447788 • Philip Harris Educational 01543 480077 • Merck BDH 0800 223344 Fungal cultures may be purchased from: • Blades Biological 01342 850242 • Philip Harris Educational 01543 480077 Notes Mouldy fruits and vegetables brought in can be used for this practical. These are likely to contain more than one organism, one of which is the causative agent and the others are opportunists, taking advantage of the decaying or dead material. There is, however, some risk with unknown Penicillium spp., as some produce penicillin. You are reminded that you should always avoid breathing in spores of unknown fungi and you are therefore advised to wear a face mask. Other suitable apple fungal pathogens which can be used include Monilinia fructigena, Nectria galigena and Botrytis cinerea. Monilinia produces a firmer but more extensive rot. Infection triggers the production of phenolics which are oxidised by host phenolases giving brown compounds which, in turn, inhibit the production of fungal pectolytic enzymes. Penicillium produces an inhibitor of the phenolases so there are fewer brown products and the pectolytic enzymes are able to break down pectins in middle lamellae of apple tissue and separate the cells, giving a paler, softer rot. This worksheet provides practical work which can help teach and illustrate many topics. These include fungal structure and nutrition; cell, tissue and plant structure; enzyme activity; decay, carbon cycle; mechanics of disease transmission, epidemiology; gene switching. From this, there are plenty of ideas for extended investigations. These practicals pose minimum risk to health, and give the opportunity to enforce good laboratory practice and the importance of aseptic technique. Useful references Ingold C T & Hudson H J (1996) The Biology of the Fungi. Chapman & Hall. ISBN 0-412-49040-4 Margulis L & Schwartz K V (1988) Five Kingdoms - 2nd edition. Freeman. ISBN 0-7167-1912-6 Webster J (1991) Introduction to Fungi. Cambridge University Press. ISBN 0-521-29699-4 Wheeler B E J (1976) An Introduction to Plant Disease. John Wiley & Sons Ltd ISBN 471-93751-7 Go to curriculum links... for more information See the
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