The Effect of Artificial Media and Temperature on the Growth and Development of the Honey Bee Brood Pathogen Ascosphaera apis

SIMPLE SUMMARY: Chalkbrood is a worldwide spread honey bee brood disease caused by the fungal pathogen Ascosphaera apis. The disease is commonly treated with fungicides, but due to the accumulation of residues, these fungicides have been banned in many countries, including European Union countries. Since then, control of chalkbrood has been problematic. The disease is fatal to individual honey bee larvae and can cause significant losses in terms of both bee numbers and colony productivity, and can even in some cases lead to colony collapse. Owing to these reasons, in vitro fungus cultivation is necessary to properly understand its pathogenesis as well as life cycle for the possible future development of an efficient and environmentally friendly control method. Therefore, in this study, several artificial media and different temperatures were evaluated to see their impact on the growth and development of A. apis. Furthermore, one of the media was modified by the addition of crushed honey bee brood to simulate natural conditions. This medium was found to be the most suitable for fungus reproductive structure production. In addition, a biological pattern was found explaining the relationships between temperature and the size of the fungal reproductive structures. ABSTRACT: Ascosphaera apis is a causative agent of chalkbrood, which is one of the most widespread honey bee diseases. In our experiments, the influence of several artificial media and cultivation under different temperatures was evaluated. Concretely, the radial growth of separated mating types was measured, reproductive structures in a Neubauer hemocytometer chamber were counted simultaneously, and the morphometry of spore cysts and spore balls was assessed. The complex set of experiments determined suitable cultivation conditions. A specific pattern between reproductive structure size and temperature was found. The optimal temperature for both mating types was 30 °C. SDA and YGPSA media are suitable for fast mycelial growth. Moreover, the effect of bee brood on fungus growth and development in vitro was investigated by modification of culture medium. The newly modified medium PDA-BB4 was most effective for the production of the reproductive structures. The result suggests that honey bee brood provides necessary nutrients for proper fungus development during in vitro cultivation. As there is no registered therapeutic agent against chalkbrood in most countries, including the European Union, the assessment of A. apis growth and development in different conditions could help to understand fungus pathogenesis and thus control chalkbrood disease.