Optimizing spore production in Chrysoporthe austraofricana for downstream transformation applications.

Griesel, J. F.*, Slupski, M., Coetzee, M. P. A., Van der Merwe, N. A.

Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa

Chrysoporthe austroafricana is a pathogenic fungus native to Southern Africa that causes stem canker in commercial Eucalyptus tree plantations. An understanding of the underlying molecular mechanisms driving pathogenicity has not been established. Thus, to further investigate genes involved in canker formation, protocols must be developed that permit targeted transformation of the pathogen. Protoplasts represent a valuable platform for the genetic transformation of fungi, since the removal of the cell wall permits insertion of a broad range of targeted genetic payloads into the cellular environment. The viability and transformational success of protoplasts rely heavily on broad factors such as tissue type or cell age. Generally, the use of germlings derived from spores when making protoplasts tends to yield higher transformation success rates, greater consistency, and greater ease of post-transformation cell wall regeneration. In the case of C. austroafricana, spore production methods are unoptimized, with pycnidia formation taking several weeks to achieve a usable number of spores for downstream experimentation. Here, methods for increasing the speed and yield of pycnidia formation were explored by testing the effects of various chemicals on growth, as well as various temperatures on pycnidia formation. It was found that a temperature of 30°C dramatically increases the yield and shortens the time of incubation needed to produce mature pycnidia. Additionally, a new method for the recovery of spores from pycnidia was established that allowed for the successful collection of a greater number of spores using a more rapid method, circumventing the need to make use of -80°C storage approaches, which would compromise downstream protoplast production.

Keywords: stem canker, pathogen, pycnidia, spores, germlings, protoplasts, Eucalyptus, temperature assay, methods, protocols