tissue infected with the spot blotch organism and wheat seed
Contact: J. Gilbert (Note: at least four weeks notice is
Cereal Research Centre
195 Dafoe Road
Winnipeg, MB R3T 2M9
Planting materials. Buy potting soil and plant pots,
or use 2 litre milk cartons cut down to approximately15 cm height.
A small (500 ml or 1 litre) garden pump sprayer
Petrie plates and potato dextrose agar (PDA). Malt agar or
Nutrient agar also may be used. Or purchase prepared agar plates.
Order from: Fisher Scientific
Tel: 1 800 234 7437
Fax: 1 800 463 2996
What to do
seedlings of wheat
Surface-sterilize infected leaf tissue
Dilute bleach 1:20 with cooled boiled water.
Immerse cut sections of leaf tissue in bleach solution for 3 mins
and then rinse in cooled boiled water.
Use alcohol or a bunsen burner to sterilize needles and loops.
Place one circle of filter paper on the bottom of a petrie plate
and another in the lid. Moisten the filter paper in the lid with
cooled boiled water. Leave the other piece dry. Place the SS leaf
tissue on the lower, dry surface. Label plates with date and ID
etc.. Put plates in plastic bags to retain humidity. Leave at room
temperature in the dark for 5 to 7 days, but check every 2-3 days
that the filter paper in the lid of the plate is still moist.
Identification of pathogen
The spores of Cochliobolus sativus, the spot blotch
pathogen are relatively large, black and shiny. They develop
either singly, or more usually, in clusters of 2 or 3. Use a
sterile needle to pick one or two spores from the infected tissue
and place in the centre of an agar plate. Expose the agar surface
for as little time as possible to prevent contamination. Label
plate with date and ID of pathogen. Place on a window sill and
keep at room temperature (20 - 23 C) for 5 to 7 days.
When the wheat seedlings are approximately 25 cm tall (21 days or
so) inoculate with a spore suspension. Wash spores from the agar
plates with cooled boiled water. Spores can be dislodged with a
sterile loop. Add one drop of detergent to reduce surface tension
and allow spores to spread on the surface of the wheat leaves. If
a haemacytometer is available you may standardize the spore
concentration to 3000 sp/ml. Otherwise add sterile water and pour
the spore suspension into the pump sprayer. Spray wheat plants
with the spore suspension until run-off. After 30 min to 1 hour
cover the plants with clear plastic bags secured around the pots
with elastic bands and leave for 24 hours. Remove bags the next
day. Leave plants on a window sill for 5 to 7 days, then examine
leaves for disease symptoms.
The final step in Koch's postulates is to re-isolate the pathogen
from the infected tissue and identify it under the microscope.
In the interests of time, the teacher may prepare the wheat plants
and inoculum ahead of time, so that students start with
inoculation and then re-isolate from the newly-infected tissue