Standard methods for fungal brood disease research

Authors: Annette Bruun Jensen, Kathrine Aronstein, José Manuel Flores, Svjetlana Vojvodic, María Alejandra Palacio, and Marla Spivak.

Table of contents

Authors
Summary
Introduction
1. Chalkbrood

   1.1. introduction
   1.2. Diagnostics and qualitative detection

      1.2.1 Morphological description
         1.2.1.1. Macroscopic diagnosis
         1.2.1.2. Microscopic diagnosis
         1.2.1.3. Biological diagnosis
      1.2.2. Molecular methods
         1.2.2.1 PCR for species identification
   1.3. Quantitative detection
      1.3.1. Quantify the level of fungal infection in bee colonies
         1.3.1.1. Procedure for quantifying the level of colony infection
         1.3.2. Molecular methods qPCR
   1.4. Production and quality of inoculums
      1.4.1. Isolation techniques
         1.4.1.1. Growth media
         1.4.1.2. Isolation of A. apis strains
         1.4.1.3. Hyphal tip isolation of A. apis strains
         1.4.1.4. Mating test
      1.4.2. PCR for strain differentiation
         1.4.2.1. PCR
      1.4.3. Preservation of in vitro cultures
         1.4.3.1. Cryopreservation in glycerol at -80°C
         1.4.3.2. Integral Rice Kernels (IRK) storage
      1.4.4. Spore production
         1.4.4.1. Production and harvest of in vitro spores
         1.4.4.2. Harvest of in vivo spores
      1.4.5. Quality test of inoculums
         1.4.5.1. Spore viability and germination test (Fig. 6)
         1.4.5.2. GLEN medium
      1.4.6. Availability and recommended reference isolates
   1.5. Infection bioassays
      1.5.1. Infection bioassays using in vitro rearing of larvae
      1.5.2. Infection bioassay of colonies
         1.5.2.1. Direct exposure of individual larvae
         1.5.2.2. Infection of colonies with a water spore suspension
         1.5.2.3. Infection of colonies with spores in pollen
   1.6. Expression of fungal genes
      1.6.1. qRT-PCR for quantification of A. apis transcripts
   1.7. Hygienic behaviour
   1.8. Olfactory detection
   1.9. Inhibitory assays against chalkbrood

      1.9.1. Inhibition of spore germination and hyphae (zone of inhibition)
      1.9.2. Inhibition in colonies
   1.10. Minimizing chalkbrood in experimental colonies
      1.10.1. Breeding for resistance
      1.10.2. Management and treatment
2. Stonebrood
   2.1. Introduction
   2.2. Biohazards
   2.3. Diagnostics and qualitative detection

      2.3.1 Morphological description
      2.3.2 Molecular methods
   2.4. Production and quality of inoculums
      2.4.1. Isolation techniques
         2.4.1.1. Aspergillus spp. can be isolated from sporulating mummies.
         2.4.1.2. Single spore isolation
      2.4.2. Preservation of in vitro cultures
   2.5. Quality test of inoculums
      2.5.1. Spore viability and germination test
      2.5.2. Availability and recommended reference isolates
   2.6. Infection bioassays
3. Future perspectives
4. References