Botryodiplodia sp. - Crops for the Future

9.1.4 Isolation of the pathogen
Materials:
• Bunsen burner
• 4 beakers
• Scalpel and forceps
• 1 empty petri-dish
• Measuring cylinder (10 ml)
• Pure PDA plates
Chemicals:
• 100 ml ethanol solution (70 %)
• Sodium hypochlorite solution (5 %)
• 250 ml distilled water
For the isolation of the pathogen partially diseased fruit are required. The fruit should have
enough healthy tissue. The fruit which were therefore bought either from local markets or
collected during the field trips.
From the fruit tissue squares were taken with the size of 1 cm² and the thickness of 3 mm. It
was important that only 1/3 of the tissue was diseased. The tools which were necessary for
this had to be sterilized after every cut (scalpel and forceps). The cuttings took place in an
empty petri-dish.
The pieces obtained were surface sterilized by dipping them
for three minutes in a 5 % sodium hypochlorite solution. The
solution was prepared immediately before the surface
sterilization, because the effectiveness of the solution
decelerates with running time.
After that the pieces were washed twice by dipping them into
Figure 23: Isoloation of the distilled water for three minutes to remove the rests of the
pathogen on rambutan (five
sterilization solution. In each case two washed fruit pieces
days old plate)
were put into one PDA plate and incubated. The mycelium of
the pathogen covered the whole plate after a few days (Figure 23) and could be subcultivated.
The pathogenicity of the isolated organism was proved using Koch’s postulates. Koch’s
postulates can be summarised in four steps (FREDRICKS and RELMAN, 1996):
• The organism must be found in all plants suffering from the disease, but not in
healthy plants.
• The organism must be isolated from a diseased plant and grown in pure culture.
• The cultured organism should cause disease when introduced into a healthy plant.
• The organism must be reisolated from the experimentally infected plant.
28