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Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species. Its impact on forest health is currently intensified, likely due to climate change, posing an increasing threat to global ecosystems and forestry. Despite extensive and successful research on this pathogen system, fundamental questions about its biology and plant-associated lifestyle remain unanswered. Addressing these questions will necessitate the development of additional experimental tools, including protocols for molecular genetics and cell biology approaches. In this study, we continue to address this need by establishing an Agrobacterium-mediated genetic transformation protocol for D. sapinea, enabling targeted mutagenesis and heterologous gene expression. We utilized this methodology to localize the histone H2B by tagging it with the fluorescent protein mCherry. Additionally, we established a time- and space-efficient laboratory-scale infection assay using two-week-old Pinus sylvestris seedlings. Integrating these tools in a proof-of-concept study enabled the visualization of D. sapinea in planta growth through the fluorescently labeled reporter strain.

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.