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When monitoring the state of health of  Fraxinus excelsior  trees, unusual symptoms were discovered within a  F. excelsior  plantation in Bosnia and Herzegovina. These symptoms included the appearance of necrosis and cankers in the basal parts of the trees, followed by the formation of fruiting bodies, however, none of these symptoms were found in the crowns. After sampling and isolation of the necrotic parts from the stem base, pathogen  Neonectria punicea  was isolated and identified from the characteristics of pure cultures, morphology of the fruiting bodies, and from multilocus sequencing. In field conditions, juvenile  F. excelsior  trees were inoculated with two  N .  punicea  isolates obtained from the necrotic tissues of both juvenile  F. excelsior  and mature  Fagus sylvatica  trees. In both isolates, 12 months post inoculation, the lengths and widths of the necroses were significantly larger compared to the control. Necroses of significantly larger lengths, widths and surfaces were found again in both tested isolates 24 months post inoculation. In the case of the  F. excelsior  isolate, the lengths of the necroses at both the stem base and at breast height increased by 1.6 times, whereas the  F. sylvatica  isolate increased in size by up to 1.7 and 1.8 times, respectively. Trees inoculated without a previous bark wound showed no symptoms, similar to the control trees. Scanning electron microscopy and X-ray micro-computed tomography imaging revealed that  N. punicea  hyphae penetrated from the cankers to the woody outermost annual growth ring and that hyphae were present mostly in the large earlywood vessels and rarely in the axial parenchyma cells. Hyphae also spread radially through the pits in vessels. The infected trees responded with the formation of tyloses in the vessels to prevent a rapid fungal spread through the axial vascular transport pathway. The ability of  N. punicea  to cause necroses in juvenile ash trees was demonstrated for the first time during this study. It poses a serious threat to planted forests and natural regenerations of  F. excelsior  especially if  F. sylvatica  is considered as a possible inoculum reservoir for future infections. This pathogen should be integrated within future ash resistance or breeding programs.

Development of Neonectria punicea Pathogenic Symptoms in Juvenile Fraxinus excelsior Trees