Large-scale ash (Fraxinus spp.) dieback caused by the fungus Hymenoscyphus fraxineus has been a major concern throughout Europe for more than two decades. Most of the related research has been focused on Fraxinus excelsior L., and there is still little information on fungal involvement in the dieback of Fraxinus angustifolia Vahl, especially in roots and stem bases, which play an important role in decline progress and tree stability. The objectives of this study were to identify fungi present in visually healthy and symptomatic wood tissues in basal parts of narrow-leaved ash trees in different decline phases, in order to determine the possible role of these fungi and their importance in the dieback process. The stem bases and roots of 90 trees in three different health categories, determined based on crown defoliation, were sampled in natural stands affected by ash dieback. Isolated fungal cultures were identified based on the rDNA ITS (Internal transcribed spacer) region and their association with tree health status was analyzed. In total, 68 different fungal taxa were confirmed, including Hymenoscyphus fraxineus and Armillaria spp., which were mainly present in roots, although overall in lower frequencies than on common ash in other studies. Most frequently isolated fungal taxa, which encompassed 51% of all obtained isolates, were Trichoderma spp., Ilyonectria robusta, Fusarium solani, Cladosporium cladosporioides, and Diaporthe cotoneastri. Their associations with tree health categories and presence in both symptomatic and visually healthy wood indicate that they act as opportunistic pathogens and early colonizers of weakened ash tissues. Research also revealed that, although the extent of crown defoliation and presence of root and stem necroses were associated, basal symptoms occurred on a number of trees with healthy looking crowns, meaning they can develop independently.

Sequoiadendron giganteum Lindl. [Buchholz] is a long-lived tree species endemic to the Sierra Nevada Mountains in California. Due to its massive size and beauty, S. giganteum is a popular ornamental tree planted in many parts of the world, including Europe. Since 2017, scattered branch die-back has been observed on S. giganteum trees in Zagreb, Croatia. Other symptoms included resinous branch cankers, reddish-brown discoloration of the sapwood and, in severe cases, crown die-back. Branches showing symptoms of die-back and cankers were collected from six S. giganteum trees in Zagreb and the aim of this study was to identify the causal agent of the disease. The constantly isolated fungi were identified using morphology and phylogenetic analyses based on the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA), and partial sequencing of two housekeeping genes, i.e., translation elongation factor 1-α (TEF 1-α), and β tubulin 2 (TUB2). The fungi were identified as Botryosphaeria dothidea (Moug.) Ces. and De Not. and Neofusicoccum yunnanense G.Q. Li & S.F. Chen. The pathogenicity test was conducted in a plant growth chamber on S. giganteum seedlings and revealed that N. yunnanense was more aggressive compared to B. dothidea. N. yunnanense was able to reproduce symptoms of canker and die-back and kill plants seven weeks after inoculation whereas B. dothidea produced cankers. To the best of our knowledge, this is the first report of B. dothidea and N. yunnanense causing canker and die-back disease of S. giganteum in Croatia. It is also the first record on the identity and pathogenicity of any fungal species associated with S. giganteum in this country. The study expended the known host range of N. yunnanense to include S. giganteum, which is a valuable ornamental tree in Croatian landscapes. Disease management strategies should be developed to mitigate or reduce the impact of the disease.

When natural regeneration of Quercus robur stands is hampered by an insufficient acorn yield, human assisted sowing of acorns collected in non-affected stands and stored for some period of time is performed. To inhibit the development of fungi and acorn deterioration during storage, thermotherapy is usually applied by submerging acorns for 2.5 h in water heated to 41 °C. This research aimed to test the effect of four thermotherapy treatments of different durations and/or applied temperatures as well as short-term storage at −1 °C or 3 °C on acorn internal mycobiota and germination. Fungal presence in cotyledons was analyzed in 450 acorns by isolation of mycelia on artificial media, followed by a DNA-based identification. Germination of 2000 acorns was monitored in an open field trial. Thermotherapy significantly decreased fungal diversity, while storage at 3 °C increased the isolation frequency of several fungi, mainly Penicillium spp. The most frequently isolated fungi did not show a negative impact on acorn germination after short-term storage. The study confirmed the efficiency of thermotherapy in the eradication of a part of acorn internal mycobiota, but also its effect on the proliferation of fast-colonizing fungi during storage. However, the latter showed to be more stimulated by storage conditions, specifically by storage at 3 °C.

Fraxinus angustifolia, the main ash species in Croatia in terms of economic and ecological importance, is affected by a severe dieback initially attributed to the fungal pathogen Hymenoscyphus fraxineus. Recently, another pathogen, Diplodia fraxini, has been shown to play a key role in ash dieback in several European countries. Therefore, because the dieback symptoms of ash trees observed in Croatia are typical of Botryosphaeriaceae attacks, the aim of this study was to define the etiology of F. angustifolia dieback. To this end, symptomatic shoots and branches and cross-sections of the main stem were sampled from 20 symptomatic trees at eight locations and analyzed for the presence of D. fraxini and other possible fungal pathogens. Diplodia fraxini was the fungus most frequently associated with branch cankers and dieback; it was isolated from 17 trees in all sites monitored, and its pathogenicity towards F. angustifolia was confirmed. The fungus was also associated with wood necrosis at the base of the trunk in two trees. Other fungi, namely H. fraxineus, Diaporthe eres, Diplodia seriata, Botryosphaeria dothidea, Armillaria gallica, and Lentinus tigrinus, were isolated sporadically.

In January 2015, an investigation of 800 ha of declining oaks in Istria and Cres (Croatian North Adriatic karst area), revealed intense dieback symptoms on Quercus cerris, Q. pubescens, and Q. ilex (Fig. 1A). Oaks make-up 82 - 100% of the trees in these stands. The disease was recorded on 357 trees/ha at the most affected site. Black stromata of various sizes were observed on branches and stems of oaks with decline symptoms (Fig. 1B-D, F, G). A larger percentage of Q. cerris and Q. pubescens trees were symptomatic than Q. ilex trees (Fig. 1E, G). Soil depth appeared to be associated with symptom severity. Oak sites revealed extensive progression of root rot and trunk decay in many trees since 2016 (Fig. 1D). Examined perithecial stromata were typical for Biscogniauxia mediterranea (BM), a causal agent of charcoal disease of oak. Collected perithecial stromata are deposited in the Herbarium of the Faculty of Forestry (UNIZG). The asci were short-stipitate, 110.5 to 169.0 × 7.8 to 12.9 µm. The ascospores we...

Narrow-leaved ash (Fraxinus angustifolia Vahl), a highly valued European forest tree species, has been severely affected by a large-scale decline, which is most probably driven by a complex of multiple interacting factors including fungi, which contribute to and accelerate this process. Red deer (Cervus elaphus L.) can be considered as one of the contributing factors, as they inflict damage on the stems of young trees by stripping the bark. These wounds not only represent suitable entry points for fungi which can cause tissue necroses and decomposition, they can lead to changes in the wood structure as well. The aims of this research were to analyze chosen parameters of bark stripping wounds in narrow-leaved ash stands, identify fungi present in the tissue exposed by wounding, and inspect the effect of wounding on the wood structure. Bark stripping was observed on ash trees from 2 cm up to 18 cm of DBH and between 0.1 m and 1.9 m of stem height. The most susceptible trees were those with an average DBH of 5 ± 2.5 cm. On most of the ash trees (51%), one-third to two-thirds of the circumference was damaged. In wounded tissue, 174 fungal isolates were found, most of which belonged to known endophytic fungi from the genera Trichoderma, Fusarium, and Clonostachys. It was observed that earlywood cells in the wounding zone had narrower lumens compared to ones in adjacent healthy zone with regard to different trees and stem heights.