1.9.1. Inhibition of spore germination and hyphae (zone of inhibition)

  1. Prepare MY20 medium (see paragraph 1.4.1.1.) in petri dishes (15 cm)
  2. Spread 2 ml of a spore solution (9.0 x 107 spores/ml) on the surface of the medium, approximately 106 spores/cm2
  3. Make a central hole (7 mm) in the MY20 medium (Fig. 7A)
  4. Place 0.5 ml of the test product into the hole
  5. Incubate the cultures (30ºC, 12 % CO2, 65 % relative humidity)
  6. Measure the fungal growth (or the zone of inhibition) (Fig. 7B)

Measure the fungal growth (or the zone of inhibition) daily using a stereoscopic microscope magnification (X 20 to 40). Mycelium growth can be seen within the medium before emerging at the surface (Puerta et al., 1990). An Image Analyzer can be used to measure the diameter of the zone of inhibition.

Fig. 7. Zone of inhibition assay of different compounds against Ascosphaera apis: A. MY-20 medium with spores spread on the entire surface and with a central hole for the test compound; B. The zone of inhibition was recorded daily by drawing on the Petri dish, a line over the border of the mycelium (each day a different colour) and the antifungal property was assessed as daily inhibition zone using an image analyser. Photos: J M Flores.

Figure 7