A genetic transformation system for Chrysoporthe austroafricana

Abstract

Chrysoporthe austroafricana is a plant pathogenic fungus that causes stem canker on Eucalyptus grandis in Southern Africa. The fungus is also known to attack non-native Tibouchina spp. and native Syzygium spp. This pathogen has been discovered in other countries such as Malawi, Mozambique, Namibia and Zambia. Despite being partially managed through breeding resistant clones, there are currently no adequate and effective control methods for this pathogen. However, no transformation methods exist for Chrysoporthe species. Therefore, it is essential to understand the role of genes associated with pathogenicity and mating of C. austroafricana by developing efficient molecular methods that could aid to increase disease resistance in Eucalyptus spp. Agrobacterium tumefaciens-mediated transformation (ATMT) has been employed successfully in several fungi over the last two decades. Many filamentous fungi such as Fusarium spp., Magnaporthe spp. and Aspergillus spp. have been transformed successfully using this transformation tool. ATMT is well known for random insertional mutagenesis in most plants and fungi. The main aim of this study was thus to develop an efficient ATMT technique for C. austroafricana, which is a prerequisite towards understanding the role of pathogenicity genes in this fungus. The Agrobacterium (AGL-1 strain) and C. austroafricana (spores) were subjected to ATMT. The highest starting spore concentration resulted in a higher number of transformants as compared to lower spore concentrations. However, overgrowth was observed on some plates, hence accurate colony counts could not be made. The transformants were resistant to hygromycin, and the green fluorescence protein (GFP) was visualized using a fluorescence microscope. The hygromycin resistance and gfp genes were amplified by polymerase chain reaction (PCR) and visualised following agarose gel electrophoresis. The number of T-DNA insert copies was determined by Southern blot analysis. All the transformants contained single insert copies at different positions, indicating that the gene markers had been randomly integrated. A few transformants with altered morphology were also observed. However, after subsequent culturing on selective and non-selective media, no morphological differences were noted. To the best of our knowledge, this is the first report of ATMT of C. austroafricana. The results obtained from this study could further help to understand the role of pathogenicity and mating-type genes of C. austroafricana through knockout studies. This information could also provide a more detailed understanding of plant-pathogen interactions between Eucalyptus and C. austroafricana. This transformation system can potentially be adapted and employed successfully in other members of Cryphonectriaceae to study the general characteristics and infection strategies of this group of plant pathogens.