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 nonwounded 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 nonwounded 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.

Diplodia Tip Blight on Its Way to the North: Drivers of Disease Emergence in Northern Europe.

Disease emergence in northern and boreal forests has been mostly due to tree-pathogen encounters lacking a co-evolutionary past. However, outbreaks involving novel interactions of the host or the pathogen with the environment have been less well documented. Following an increase of records in Northern Europe, the first large outbreak of Diplodia sapinea on Pinus sylvestris was discovered in Sweden in 2016. By reconstructing the development of the epidemic, we found that the attacks started approx. 10 years back from several isolated trees in the stand and ended up affecting almost 90% of the trees in 2016. Limited damage was observed in other plantations in the surroundings of the affected stand, pointing to a new introduced pathogen as the cause of the outbreak. Nevertheless, no genetic differences based on SSR markers were found between isolates of the outbreak area and other Swedish isolates predating the outbreak or from other populations in Europe and Asia Minor. On a temporal scale, we saw that warm May and June temperatures were associated with higher damage and low tree growth, while cold and rainy conditions seemed to favor growth and deter disease. At a spatial scale, we saw that spread occurred predominantly in the SW aspect-area of the stand. Within that area and based on tree-ring and isotope (δ13C) analyses, we saw that disease occurred on trees that over the years had shown a lower water-use efficiency (WUE). Spore traps showed that highly infected trees were those producing the largest amount of inoculum. D. sapinea impaired latewood growth and reduced C reserves in needles and branches. D. sapinea attacks can cause serious economic damage by killing new shoots, disrupting the crown, and affecting the quality of stems. Our results show that D. sapinea has no limitations in becoming a serious pathogen in Northern Europe. Management should focus on reducing inoculum, especially since climate change may bring more favorable conditions for this pathogen. Seedlings for planting should be carefully inspected as D. sapinea may be present in a latent stage in asymptomatic tissues.