1.4.1.4. Mating test

Once a single isolate is obtained, the mating type can be determined using a sexual compatibility test. We recommend that the two reference strains (AFSEF 7405 and ARSEF 7406) be used in the mating tests.

  1. Cut agar plugs from the border of well growing isolates
  2. Place the plugs of the new isolate between agar plugs of the two reference strains with a distance of approximately 4 cm
  3. Incubate the plate in darkness at 30-34 °C
  4. After 5-7 days the presence of dark spore cysts (Fig. 5) normally can be observed as a black zone where hyphae of two strains with opposite mating types meet, indicating successful reproduction
  5. If the test isolates are sexually compatible with the reference + strain, the other is the – mating type, or vice versa

On a standard agar plate (diameter 9 cm) 4 new isolates can be tested simultaneously (Fig. 5).

Fig. 5. Ascosphaera apis mating test and production of inoculums. A. Mating test with four isolates of unknown mating types. Unknown 1 mates with ARSEF 7405 (+) and therefore harbours the opposite mating type (-). B. Production of inoculums between two isolates of opposite mating types. Fruiting bodies with spores inside are formed where the hyphae meet (black zone). Photo: A B Jensen.

Figure 5