A reliable and simple method for the production of viable pycnidiospores of the pine pathogen Diplodia sapinea and a spore-based infection assay on Scots pine

Diplodia sapinea is a globally distributed opportunistic fungal pathogen of conifers that causes severe production losses in forestry. The fungus frequently colonizes pine trees as an endophyte without causing visible symptoms but can become pathogenic when the host plant is weakened by stress, such as drought or heat. Forest damage might therefore further increase due to the effects of climate change. The future development of control strategies depends on a better understanding of the fungus´ biology, which requires experimental methods for its investigation in the laboratory. An efficient, standardized protocol for the production and storage of highly viable pycnidiospores was developed, and a spore-based infection method was devised. We compared infection rates of dormant and actively growing, wounded or non-wounded Scots pine seedlings inoculated with in vitro produced spores and mycelium from agar-plugs. Spores were a much more efficient inoculum for causing disease symptoms on wounded plants than the conventional agar plug. The application of spores on non-wounded plants lead to high rates of asymptomatic infection, suggesting endophytic fungal development. These methods enable standardized spore infection and virulence assays and promote D. sapinea as a model organism for studying the switch from endophytic to pathogenic life styles of forest pathogens.